Method of treatment and prophylaxis of venous thrombosis

ABSTRACT

The invention relates to new competitive inhibitors of thrombin, their synthesis, pharmaceutical compositions containing the compounds as active ingredients, and the use of the compounds as anticoagulants for prophylaxis and treatment of thromboembolic diseases, according to the formula ##STR1## wherein A represents a methylene group, an ethylene group or a propylene group, which may be substituted or 
     A represents --CH 2  --O--CH 2  --, --CH 2  --S--CH 2  --, --CH 2  --SO--CH 2  --, or 
     A represents --CH 2  --O--, --CH 2  --S--, --CH 2  --SO--, with the heteroatom functionality in position 4, or 
     n is an integer 2 to 6; and 
     B represents --N(R 6 )--C(NH)--NH 2 , wherein R 6  is H or a methyl group, or 
     B represents --S--C(NH)--NH 2 , or --C(NH)--NH 2 . 
     Further described is new use in synthesis of pharmaceutical compounds of a compound of the formula: ##STR2##

This application is a continuation of application Ser. No. 07/984,884,filed on Dec. 2, 1992, now U.S. Pat. No. 5,614,499.

This invention relates to new competitive inhibitors of thrombin, theirsynthesis, pharmaceutical compositions containing the compounds asactive ingredients, and the use of the compounds as anticoagulants forprophylaxis and treatment of thromboembolic diseases such as venousthrombosis, pulmonary embolism, arterial thrombosis, in particularmyocardial infarction and cerebral thrombosis, general hypercoagulablestates and local hypercoagulable states, e.g. following angioplasty andcoronary bypass operations.

The invention also relates to novel use of a compound as a startingmaterial in synthesis of a serine protease inhibitor. Furthermore theinvention relates to a novel structural fragment in a serine proteaseinhibitor.

BACKGROUND

Blood coagulation is the key process involved in both haemostasis (i.e.prevention of blood loss from a damaged vessel) and thrombosis (i.e. thepathological occlusion of a blood vessel by a blood clot). Coagulationis the result of a complex series of enzymatic reactions, where one ofthe final steps is conversion of the proenzyme prothrombin to the activeenzyme thrombin.

Thrombin plays a central role in coagulation. It activates platelets, itconverts fibrinogen into fibrin monomers, which polymerise spontaneouslyinto filaments, and it activates factor XIII, which in turn crosslinksthe polymer to insoluble fibrin. Thrombin further activates factor V andfactor VIII in a positive feedback reaction. Inhibitors of thrombin aretherefore expected to be effective anticoagulants inhibition ofplatelets, fibrin formation and fibrin stabilization. By inhibiting thepositive feedback mechanism they are expected to excert inhibition earlyin the chain of events leading to coagulation and thrombosis.

PRIOR ART

Inhibitors of thrombin based on the amino acid sequence around thecleavage site for the fibrinogen Aα chain were first reported byBlomback et al in J. Clin. Lab. Invest. 24, suppl 107, 59, (1969), whosuggested the sequence Phe-Val-Arg (P9-P2-P1, herein referred to as theP3-P2-P1 sequence) to be the best inhibitor.

In U.S. Pat. No. 4,346,078 (Richter Gedeon Vegyeszeti Gyar R. T.,priority date Oct. 7, 1980) and in Peptides 1983 by Walter de Gruyter &Co, Berlin, pp 643-647, S. Bajusz et al described the thrombin inhibitorH-DPhe-Pro-Agm, a dipeptidyl derivative with an aminoalkyl guanidine inthe P1-position.

S. Bajusz et. al. also reported in J. Med. Chem. 1990, 33, 1729-1735 andin EP-A2-0,185,390 (Richter Gedeon Vegyeszeti Gyar R. T.) (priority dateDec. 21, 1984) that replacing the agmatine with an arginine aldehydegave a thrombin inhibitor which had much higher potency.

The reason for the increased activity of this thrombin inhibitor isthought possibly to be due to interaction of the aldehyde function withthe Ser-OH in the active site of the enzyme forming a hemiacetal. It isnot concievable to have the same type of interaction in the dipetidederivative H-DPhe-Pro-Agm since it does not have an amino acidderivative with a carbonyl group in the P1-position.

In other work in the thrombin inhibitor field, inhibitors of serineproteases that are based on electrophilic ketones instead of aldehydesin the P1-position include the following:

E. N. Shaw et al. (Research Corporation) U.S. Pat. No. 4,318,904(priority date Apr. 25, 1980) describing peptide chloro-methyl ketonese.g. H-DPhe-Pro-Arg-CH₂ Cl.

M. Szelke and D. M. Jones in EP-A1-0,118,280, (priority date Mar. 4,1983) describing compounds derived from the P₃ -P₂ ' pentapeptidesequence of the fibrinogen Aα chain in which the scissile P₁ -P₁ 'peptide bond was replaced with the --CO--CH₂ -moiety, forming a ketoisostere to the corresponding peptides.

M. Kolb et. al. (Merrell-Dow) EP-A2-0,195,212 (Priority date Feb. 4,1985) describing peptidyl α-keto esters and amides.

B. Imperiali and R. H. Abeles, Biochemistry 1986. 25. 3760 describingpeptidyl fluoroalkyl ketones.

D. Schirlin et al. (Merrell-Dow) EP-A1-0,362,002 (priority date Sep. 1,1988) describing fluoroalkylamide ketones.

P. Bey et al., (Merrell-Dow) EP-A2-0,364,344 (priority date Sep. 1,1988) describing α,β,δ- triketo compounds.

Ueda et al., Biochem. J. 1990, 265, 539 also describing peptidylfluoroalkyl ketones.

Inhibitors of thrombin based on C-terminal boronic acid derivatives ofarginine and isothiouronium analogues thereof have been reported by A. DKettner et al. (Du Pont) EP-A2-0,293,881 (priority dates Jun. 5, 1987and Apr. 6, 1988).

An object of the present invention is to provide novel and potentthrombin inhibitors with competitive inhibitory activity towards theirenzyme i.e. causing reversible inhibition. A further object is to obtaininhibitors which are orally bioavailable and selective inhibitingthrombin over other serine proteases. Stability, duration of action, andlow toxicity at therapeutic dosages are still further objects of theinvention.

DISCLOSURE OF THE INVENTION

Compounds

Compounds of the invention relate to the peptide sequence of humanfibrinogen Aa chain representing modified sub-sites P₉. P₂ and P₁ :##STR3## The above compound is identified as SEQ ID NO:1 in the SequenceListing.

According to the invention it has been found that compounds of thegeneral Formula I, either as such or in the form of physiologicallyacceptable salts, and including stereoisomers, are potent inhibitors ofthrombin: ##STR4## wherein: A represents a methylene group, or

A represents an ethylene group and the resulting 5-membered ring may ormay not carry one or two fluorine atoms, a hydroxy group or an oxo groupin position 4, or may or may not be unsaturated, or

A represents --CH₂ --O--, --CH₂ --S--, --CH₂ --SO--, with the heteroatomfunctionality in position 4, or

A represents a n-propylene group and the resulting 6-membered ring mayor may not carry in position 5 one fluorine atom, a hydroxy group or anoxo group, carry two fluorine atoms in one of positions 4 or 5 or beunsaturated in position 4 and 5, or carry in position 4 an alkyl groupwith 1 to 4 carbon atoms, or

A represents --CH₂ --O--CH₂ --, --CH₂ --S--CH₂ --, --CH₂ --SO--CH₂ --;

R¹ represents H, an alkyl group having 1 to 4 carbon atoms, ahydroxyalkyl group having 2-3 carbon atoms or R¹¹ OOC-alkyl-, where thealkyl group has 1 to 4 carbon atoms and R¹¹ is H or an alkyl grouphaving 1 to 4 carbon atoms or an alkylene group having 2-3 carbon atomsintramolecularly bound alpha to the carbonyl group in R¹, or

R¹ represents R¹² OOC-1,4-phenyl-CH₂ --, where R¹² is H or an alkylgroup having 1 to 4 carbon atoms, or

R¹ represents R¹³ --NH--CO-alkyl-, where the alkyl group has 1 to 4carbon atoms and is possibly substituted alpha to the carbonyl with analkyl group having 1 to 4 carbon atoms and where R¹³ is H or an alkylgroup having 1 to 4 carbon atoms or --CH₂ COOR¹² where R¹² is as definedabove, or

R¹ represents R¹² OOC--CH₂ --OOC-alkyl-, where the alkyl group has 1 to4 carbon atoms and is possibly substituted alpha to the carbonyl with analkyl group having 1 to 4 carbon atoms and where R¹² is as definedabove, or

R¹ represents CH₃ SO₂ --, or

R¹ represents R¹² OCOCO-- where R¹² is as defined above, or

R¹ represents --CH₂ PO(OR¹⁴)₂, --CH₂ SO₃ H or --CH₂ -(5-(1H)-tetrazolyl)where R¹⁴ is, individually at each occurrence, H, methyl or ethyl;

R² represents H or an alkyl group having 1 to 4 carbon atoms or R²¹OOC-alkyl-, where the alkyl group has 1 to 4 carbon atoms and ispossibly substituted in the position which is alpha to the carbonylgroup, and the alpha substituent is a group R²² --(CH₂)_(p) --, whereinp=0-2 and R²² is methyl, phenyl, OH, COOR²¹, and R²¹ is H or an alkylgroup having 1 to 4 carbon atoms.

m is 0, 1 or 2, R³ represents a cyclohexyl group and R⁴ represents H, or

m is 1 and R³ represents a cyclohexyl or phenyl group and R⁴ forms anethylene bridge together with R¹, or

m is 1 and R³ and R⁴ each represents a cyclohexyl or phenyl group;

R⁵ represents H or an alkyl group having 1 to 4 carbon atoms;

n is an integer 2 to 6; and

B represents --N(R⁶)--C(NH)--NH₂, wherein R⁶ is H or a methyl group, or

B represents --S--C(NH)--NH₂, or --C(NH)--NH₂.

An alkyl group may be straight or branched unless specified otherwise.Alkyl groups having 1 to 4 carbon atoms are methyl, ethyl, n-propyl,i-propyl, n-butyl, i-butyl, s-butyl and t-butyl. When unsaturation isreferred to, a carbon-carbon double bond is intended. Abbreviations arelisted at the end of this specification.

According to a preferred embodiment the invention relates to compoundsof Formula I, wherein R¹ represents R¹¹ OOC-alkyl-, where the alkylgroup has 1 to 4 carbon atoms and R¹¹ is H. Of those compounds, thecompounds where A is ethylene and R⁵ is H or an alkyl group having 1 to4 carbon atoms, particularly those where R⁵ is H are preferred.

Of the compound of Formula I, those compounds where R³ is cyclohexyl, mis 1 or 2, particularly m is 1 and R⁴ is H constitute another preferredsubclass.

Another preferred group of compounds are the compounds where A isn-propylene and the resulting 6-membered ring may or may not carry inposition 4 an alkyl group with 1 to 4 carbon atoms, and R⁵ is H or analkyl group having 1 to 4 carbon atoms, particularly those where R⁵ isH.

According to another preferred embodiment n is 3.

Compounds of Formula I having S-konfiguration on the α-amino acid in theP2-position are preferred ones, of those compounds also havingR-konfiguration on the α-amino acid in the P3-position are particularlypreferred ones.

Preferred compounds of the invention are:

    ______________________________________                                        Example No.                                                                           Compound                                                              ______________________________________                                         1      H--(R)Cha-Pro-Agm                                                      2      Me-(R)Cha-Pro-Agm                                                      3      HO--(CH.sub.2).sub.3 --(R)Cha-Pro-Agm                                  4      HOOC--CH.sub.2 --(R)Cha-Pro-Agm                                        5      .sup.i PrOOC--CH.sub.2 --(R)Cha-Pro-Agm                                6      HOOC--CH.sub.2 -(Me)(R)Cha-Pro-Agm                                     7      HOOC--(R,S)CH(Me)-(R)Cha-Pro-Agm                                       8      HOOC--(RorS)CH(Me)-(R)Cha-Pro-Agm/a                                    9      HOOC--(RorS)CH(Me)-(R)Cha-Pro-Agm/b                                   10      HOOC--(RorS)CH(.sup.n Pr)-(R)Cha-Pro-Agm/a                            11      HOOC--(RorS)CH(.sup.n Pr)-(R)Cha-Pro-Agm/b                            12      HOOC--(RorS)CH(Ph)-(R)Cha-Pro-Agm/b                                   13      HOOC--(R,S)CH(CH.sub.2 CH.sub.2 Ph)-(R)Cha-Pro-Agm                    14      HOOC--(RorS)CH(CH.sub.2 CH.sub.2 Ph)-(R)Cha-Pro-Agm/a                 15      HOCC--CH.sub.2 -CH.sub.2 --(R)Cha-Pro-Agm                             16      EtCOC--CO--(R)Cha-Pro-Agm                                             17      (R,S)Bla-(R)Cha-Pro-Agm                                               18      HOOC--(RorS)CH(CH.sub.2 CH.sub.2 Ph)-(R)Cha-Pro-Agm/b                 19      H--(R)Cha-Pro-Nag                                                     20      .sup.n Bu-(R)Cha-Pro-Nag                                              21      HO-(CH.sub.2).sub.3 --(R)Cha-Pro-Nag                                  22      HOOC--CH.sub.2 --(R)Cha-Pro-Nag                                       23      EtOOC--CH.sub.2 --(R)Cha-Pro-Nag                                      24      .sup.n PrOOC--CH.sub.2 --(R)Cha-Pro-Nag                               25      .sup.t BuOOC--CH.sub.2 --(R)Cha-Pro-Nag                               26      HOOC--CH.sub.2 -OOC--CH.sub.2 --(R)Cha-Pro-Nag                        27      H.sub.2 N-CO-CH.sub.2 --(R)Cha-Pro-Nag                                28      HOOC--CH.sub.2 FNH-CO-CH.sub.2 --(R)Cha-Pro-Nag                       29      (HOOC--CH.sub.2).sub.2 --(R)Cha-Pro-Nag                               30      HOOC--CH.sub.2 -(Me)(R)Cha-Pro-Nag                                    31      HOOC--CH.sub.2 -(nBu)(R)Cha-Pro-Nag                                   32      HCOC--(R,S)CH(Me)-(R)Cha-Pro-Nag                                      33      HOOC--(RorS)CH(Me)-(R)Cha-Pro-Nag/a                                   34      HOCC--(RorS)CH(Me)-(R)Cha-Pro-Nag/b                                   35      EtOOC--(R,S)CH(Me)-(R)Cha-Pro-Nag                                     36      .sup.n HOOC--(RorS)CH(.sup.n Pr)--(R)Cha-Pro-Nag/a                    37      HOOC--(R)CH(CH.sub.2 -OH)--(R)Cha-pro-Nag                             38      HOOC--(R,S)CH(Ph)-(R)Cha-Pro-Nag                                      39      HOOC-(S)CH(CH.sub.2 CH.sub.2 Ph)-(R)Cha-Pro-Nag                       40      HOOC--(R)CH(CH.sub.2 CH.sub.2 Ph)-(R)Cha-Pro-Nag                      41      HOOC--CH.sub.2 -CH.sub.2 --(R)Cha-Pro-Nag                             42      EtOOC--CH.sub.2 -CH.sub.2 --(R)Cha-Pro-Nag                            43      HOOC-(CH.sub.2).sub.3 --(R)Cha-Pro-Nag                                44      EtOOC-(CH.sub.2).sub.3 --(R)Cha-Pro-Nag                               45      HOOC--CO--(R)Cha-Pro-Nag                                              46      MeOOC--CO--(R)Cha-Pro-Nag                                             47      (R,S)Bla-(R)Cha-Pro-Nag                                               48      HOOC--(R,S)CH(CH.sub.2 COOH)--(R)Cha-Pro-Nag                          49      MeOOC--(R,S)CH(CH.sub.2 COOMe)-(R)Cha-Pro-Nag                         50      HOOC-Ph-4-CH.sub.2 --(R)Cha-Pro-Nag                                   51      (HO).sub.2 P(O)-CH.sub.2 --(R)Cha-Pro-Nag                             52      EtO(HO)P(O)-CH.sub.2 --(R)Cha-Pro-Nag                                 53      (EtO).sub.2 P(O)-CH.sub.2 --(R)Cha-Pro-Nag                            54      HOOC--CH.sub.2 --(R)Cha-Pro-Mag                                       55      H--(R,S)Pro(3-Ph)-Pro-Agm                                             56      H--(R,S)Pro(3-(trans)Ch)-Pro-Agm                                      57      HOOC--CH.sub.2 --(R,S)Pro(3-(trans)Ph)-Pro-Agm                        58      HOOC--CH.sub.2 --(R,S)Pro(3-(trans)Ph)-Pro-Nag                        59      HOOC--CH.sub.2 --(R)Cha-Pic-Agm                                       60      HOOC--CH.sub.2 -(Me)(R)Cha-(R,S)Pic-Agm                               61      HOOC--(R,S)CH(Me)-(R)Cha-Pic-Agm                                      62      HOOC--(RorS)CH(Me)-(R)Cha-Pic-Agm/a                                   63      HOOC--(RorS)CH(Me)-(R)Cha-Pic-Agm/b                                   64      HOOC--CH.sub.2 -CH.sub.2 --(R)Cha-Pic-Agm                             65      H--(R)Cha-Pic-Nag                                                     66      Me-(R)Cha-(R,S)Pic-Nag                                                67      HOOC--CH.sub.2 --(R)Cha-Pic-Nag                                       68      MeOOC--CH.sub.2 --(R)Cha-Pic-Nag                                      69      .sup.i PrOOC--CH.sub.2 --(R)Cha-Pic-Nag                               70      HOOC--CH.sub.2 -(Me)(R)Cha-(RorS)Pic-Nag/b                            71      HOOC--(R,S)CH(Me)-(R)Cha-(R,S)Pic-Nag                                 72      HOOC--(RorS)CH(Me)-(R)Cha-(RorS)Pic-Nag/c                             73      HOOC--(RorS)CH(Me)-(R)Cha-(RorS)Pic-Nag/d                             74      HOOC--CH.sub.2 -CH.sub.2 --(R)Cha-Pic-Nag                             75      HOOC--CH.sub.2 --(R)Cha-(R,S)Mor-Agm                                  76      HOOC--CH.sub.2 --(R)Cha-(RorS)Mor-Nag                                 77      H--(R)Cha-Aze-Nag                                                     78      HOOC--CH.sub.2 --(R)Cha-Aze-Nag                                       79      H--(R)Cha-Pro(5-(S)Me)-Nag                                            80      HOOC--CH.sub.2 --(R)Cha-Pro(5-(S)Me)-Nag                              81      HOOC--CH.sub.2 --(R)CHa-(RorS)Pic(4,5-dehydro)-Nag/b                  82      HOOC--CH.sub.2 --(R)Cha-Pic(4-(S)Me)-Nag                              83      HOOC--CH.sub.2 --(R)Cha(R)Pic(4(R)Me)-Nag                             84      HOOC--CH.sub.2 --(R)Cgl-Pic-Nag                                       85      H--(R)Hoc-Pro-Nag                                                     86      HOOC--CH.sub.2 --(R)Hoc-Pro-Nag                                       87      HOOC--CH.sub.2 --(R)Hoc-Pic-Nag                                       88      HOOC--CH.sub.2 --(R)Dph-Pic-Nag                                       89      HOOC--CH.sub.2 --(R)Dch-Pic-Nag                                       90      HOOC--CH.sub.2 --(R)Cha-Pro(5-(R,S)Me)-Nag                            91      H--(R)Cha-Pic(4-(R)Me)-Nag                                            92      HOOC--CH.sub.2 --(R)Cha-Pic(4-(R)Me)-Nag                              93      HOOC--CH.sub.2 --(R)Cha-Pic(6-(S)Me)-Nag                              ______________________________________                                    

Of those compounds, the compounds having Example Nos. 4, 6, 9, 22, 30,34, 59, 63, 67, 73, 80 and 82 are particularly preferred, and of thosethe following compounds are most preferred:

    ______________________________________                                        30         HOOC--CH.sub.2 -(Me)(R)Cha-Pro-Nag                                 34         HOOC--(RorS)CH(Me)-(R)Cha-Pro-Nag/b                                67         HOOC--CH.sub.2 -(R)Cha-Pic-Nag                                     ______________________________________                                    

The most preferred compound among compounds of Formula I is HOOC--CH₂(R)Cha-Pic-Nag.

In the above tables of compounds, the letters /a, /b, /c and /d refer toa substantially pure stereoisomer at the carbon atom denoted "RorS". Thestereoisomer can be identified for each compound with reference to theexperimental part herein. "R,S" refers to a mixture of stereoisomers.

In a further embodiment the invention relates to novel use of a compoundof the formula: ##STR5## as a starting material in synthesis of a serineprotease inhibitor, and in particular in synthesis of a thrombininhibitor. It can be used as such or having the guanidino group eithermono protected at the δ-nitrogen or diprotected at the δ-nitrogens orthe γ, δ-nitrogens, preferably with a protective group such as benzyloxycarbonyl. Protection of the noragmatine derivatives is carried out bymethods known in the art for guanidino compounds. This compound is named"noragmatine" or "Nag" herein. The compound has been previouslydisclosed inter alia as a hair bleaching accelerator in GB 1,599,324(Henkel, priority date Feb. 5, 1977). The structural fragment of theformula ##STR6## has however not been previously disclosed as astructural element in a pharmaceutically active compound. As suchstructural element the "noragmatine" fragment renders a serine proteaseinhibitor, and in particular a thrombin inhibitor valuable.

Medical and pharmaceutical use

In a further embodiment the invention relates to treatment, in a humanor animal organism, of conditions where inhibition of thrombin isrequired. The compounds of the invention are expected to be useful inparticular in animals including man in treatment or prophylaxis ofthrombosis and hypercoagulability in blood and tissues. It isfurthermore expected to be useful in situations where there is anundesirable excess of the thrombin without signes of hypercoagulability.Disease states in which the compounds have a potential utility, intreatment and/or prophylaxis, include venous thrombosis and pulmonaryembolism, arterial thrombosis, such as in myocardial infarction,unstable angina, thrombosis-based stroke and peripheral arterialthrombosis. Further, the compounds have expected utility in prophylaxisof atherosclerotic diseases such as coronary arterial disease, cerebralarterial disease and peripheral arterial disease. Further, the compoundsare expected to be useful together with thrombolytics in thromboticdiseases, in particular myocardial infarction. Further, the compoundshave expected utility in prophylaxis for re-occlusion afterthrombolysis, percutaneous trans-luminal angioplasty (PTCA) and coronarybypass operations. Further, the compounds have expected utility inprevention of re-thrombosis after microsurgery. Further, the compoundsare expected to be useful in anticoagulant treatment in connection withartificial organs and cardiac valves. Further, the compounds haveexpected utility in anticoagulant treatment in haemodialysis anddisseminated intravascular coagulation.

A further expected utility is in rinsing of catheters and mechanicaldevises used in patients in vivo, and as an anticoagulant forpreservation of blood, plasma and other blood products in vitro.

Pharmaceutical preparations

The compounds of the Formula I will normally be administered by theoral, rectal, dermal, nasal or parenteral route in the form ofpharmaceutical preparations comprising the active ingredient either as afree base or a pharmaceutical acceptable non-toxic acid addition salt,e.g. the hydrochloride, hydrobromide, lactate, acetate, citrate,p-toluenesulfonate, trifluoroacetate and the like in a pharmaceuticallyacceptable dosage form.

The dosage form may be a solid, semisolid or liquid preparation preparedby per se known techniques. Usually the active substance will constitutebetween 0.1 and 99% by weight of the preparation, more specificallybetween 0.1 and 50% by weight for preparations intended for parenteraladministration and between 0.2 and 75% by weight for preparationssuitable for oral administration.

Suitable daily doses of the compounds of the invention in therapeuticaltreatment of humans are about 0.001-100 mg/kg body weight at peroraladministration and 0.001-50 mg/kg body weight at parenteraladministration.

Preparation

A further objective of the invention is the mode of preparation of thecompounds. The compounds of Formula I may be prepared by coupling of anN-terminally protected amino acid or dipeptide or a preformed,N-terminally alkylated protected dipeptide to a compound

    H.sub.2 N--(CH.sub.2).sub.n --X

wherein n is as defined with Formula I and X is an unprotected orprotected guanidino group or a protected amino group, or a grouptransferable into an amino group, where the amino group is subsequentlytransferred into a guanidino group.

The coupling is accordingly done by one of the following methods:

Method I

Coupling of an N-terminally protected dipeptide, prepared by standardpeptide coupling, with either a protected- or unprotected aminoguanidine or a straight chain alkylamine carrying a protected or maskedamino group at the terminal end of the alkyl chain, using standardpeptide coupling, shown in the formula ##STR7## wherein R³, R⁴, R⁵, n, mand A are as defined in Formula I, R⁶ is H or alkyl, W₁ is an aminoprotecting group such as tertiarybutoxy carbonyl and benzyloxy carbonyland X is --NH--C(NH)NH₂, --NH--C(NH)NH--W₂, --N(W₂)--C(NH)NH--W₂,--NH--C(NW₂)NH--W₂ or --NH--W₂, where W₂ is an amine protecting groupsuch as tertiarybutoxy carbonyl or benzyloxy carbonyl, or X is a maskedamino group such as azide, giving the protected peptide. The finalcompounds can be made in any of the following ways, depending on thenature of the X-group used: Removal of the protecting group(s) (whenX=--NH--C(NH)NH₂, --N(W₂)--C(NH)NH--W₂, --NH--C(NW₂)NH--W₂ or--NH--C(NH)NH--W₂), or a selective deprotection of the W₁ -group (e.gwhen X=--NH--C(NH)NH--W₂, --N(W₂)--C(NH)NH--W₂, --NH--C(NW₂)NH--W₂, W₂in this case must be orthogonal to W₁) followed by alkylation of theN-terminal nitrogen and deprotection or a selectivedeprotection/unmasking of the terminal alkylamino function (X=NH--W₂, W₂in this case must be orthogonal to W₁ or X=a masked aminogroup, such asazide) followed by a guanidation reaction, using standard methods, ofthe free amine and deprotection of the W₁ -group.

Method II

Coupling of an N-terminally protected amino acid, prepared by standardmethods, with either a protected- or unprotected amino guanidine or astraight chain alkylamine carrying a protected or masked amino group atthe terminal end of the alkyl chain, using standard peptide coupling,shown in the formula ##STR8## wherein W₁, A, R⁵ and X are as definedabove followed by deprotection of the W₁ -group and coupling with theN-terminal amino acid, in a protected form, leading to the protectedpeptide described in Method I or III, depending on the choice of thesubstitution pattern on the nitrogen of the N-terminal amino acid usedin the coupling. The synthesis is then continued according to Method Ior Method III to give the final peptides.

Method III

Coupling of a preformed N-terminally alkylated and protected dipeptide,prepared by standard peptide coupling, with either a protected orunprotected amino guanidine or a straight chain alkylamine carrying aprotected or masked aminogroup at the terminal end of the alkyl chain,using standard peptide coupling, shown in the formula ##STR9## whereinR², R³, R⁴, R⁵, n, m, A and X are defined as above provided that R² isother than H and W₃ is an acyl protecting group such as trifluoroacyl.

The final compounds can be made in any of the following ways dependingon the nature of the X-group used: Removal of protecting groups (when X=NH--C(NH)NH₂, NH--C(NH)NH-W₂, N(W₂)--C(NH)NH--W₂, NH--C(NW₂)NH--W₂ orNH--W₂) or a selective deprotection/unmasking of the terminal alkylaminofunction (X=NH--W₂, W₂ in this case must be orthogonal to W₃ or X=amasked amino group such as azide) followed by a guanidation deprotectionof the W₃ group

DETAILED DESCRIPTION OF THE INVENTION

The following description is illustrative of aspects of the invention.

EXPERIMENTAL PART

Synthesis of the compounds of the invention is illustrated in Schemes Ito VI appended hereto.

General Experimental Procedures

The ¹ H NMR and ¹³ C NMR measurements were performed on BRUKER AC-P 300and BRUKER AM 500 spectrometers, the former operating at a ¹ H frequencyof 500.14 MHz and a ¹³ C frequency of 125.76 MHz and the latter at ¹ Hand ¹³ C frequency of 300.13 MHz and 75.46 MHz respectively.

The samples were 10-50 mg dissolved in 0.6 ml of either of the followingsolvents; CDCl₃ (isotopic purity>99.8%, Dr. Glaser A. G. Basel), CD₃ OD(isotopic purity>99.95%, Dr. Glaser A. G. Basel) or D₂ O (isotopicpurity>99.98%, Dr. Glaser A. G. Basel).

The ¹ H and ¹³ C chemical shift values in CDCl₃ and CD₃ OD are relativeto tetramethylsilane as an external standard. The ¹ H chemical shifts inD₂ O are relative to the sodium salt of 3-(trimethylsilyl)-d₄ -propanoicacid and the ¹³ C chemical shifts in D₂ O are referenced relative to1,4-dioxane (67.3 ppm), both as external standard. Calibrating with anexternal standard may in some cases cause minor shift differencescompared to an internal standard, however, the difference in ¹ Hchemical shift is less than 0.02 ppm and in ¹³ C less than 0.1 ppm.

The ¹ H NMR spectrum of peptide sequences containing a proline residuefrequently exhibits two sets of resonances. This corresponds to theexistence of to contributing conformers with respect to the rotationaround the amide bond, where proline is the N-part of the amide bond.The conformers are named cis and trans. In our compounds the sequences(R)Cha-Pro- and -(R)Cha-Pic- often give rise to a cis-trans equilibriumwith one conformer as the preponderant conformer (>90%). In those casesonly the ¹ H chemical shifts of the major rotamer is reported.

Thin-Layer Chromatography was carried out on commercial Merck Silicagel60F₂₅₄ coated glass or aluminium plates. Visualization was by acombination of UV-light, followed by spraying with a solution preparedby mixing 372 ml of EtOH(95%), 13.8 ml of concentrated H₂ SO₄, 4.2 ml ofconcentrated acetic acid and 10.2 ml of p-methoxy benzaldehyde orphosphomolybdic acid reagent (5-10 w.t % in EtOH(95%)) and heating.

Flash chromatography was carried out on Merck Silicagel 60 (40-63 mm,230-400 mesh) under pressure of N₂.

Reversed phase high-performance liquid chromatography (in the Examplesreferred to as RPLC) was performed on a Waters M-590 instrument equippedwith three reverse phase Kromasil 100, C8 columns (Eka-Nobel) havingdifferent dimensions for analytical (4.6 mm×250 mm), semipreparative(1"×250 mm) and preparative (2"×500 mm) chromatography detecting at 226nm.

Freeze-drying was done on a Leybold-Heraeus, model Lyovac GT 2,apparatus.

Protection Procedures

Boc-(R)Cha-OH

To a solution of H-(R)Cha-OH, 21.55 g (125.8 mmol), in 130 ml 1M NaOHand 65 ml THF was added 30 g (137.5 mmol) of (Boc)₂ O and the mixturewas stirred for 4.5 h at room temperature. The THF was evaporated and anadditional 150 ml of water was added. The alkaline aqueous phase waswashed twice with EtOAc, then acidified with 2M KHSO₄ and extracted with3×150 ml of EtOAc. The combined organic phase was washed with water,brine and dried (Na₂ SO₄). Evaporation of the solvent afforded 30.9 g(90.5%) of the title compound as a white solid.

Z-(R)Cha-OH

The same procedure as described in Bodanszky M. and Bodanszky A. "ThePractice of Peptide Synthesis", Springer-Verlag, 1984, p. 12, was usedstarting from H-(R)Cha-OH.

Boc-(Me)Phe-OH

Prepared in the same way as Boc-(R)Cha-OH from Me-(R)Phe-OH.

Boc-(R,S)Pro(3-(trans)Ph)-OH

To a well stirred solution of 2.0 g (8.8 mmol, 1 eq.)H-(R,S)Pro(3-(trans)Ph)-OH×HCl (Prepared as described in J. Org. Chem.,55, p. 270-75 , 1990 and J. Org. Chem., 39, 1710-1716, 1974), in 17.6 mlof 1N NaOH, 12 ml of H₂ O and 12 ml of THF at +5° C. was added 2.33 g(Boc)₂ O (10.7 mmol, 1.2 eq.). The reaction was allowed to reach roomtemperature and the stirring was continued for an additional 18 h. Theorganic solvent was evaporated and 50 ml of H₂ O was added to theresidue. The basic water phase was washed with 2×50 ml of EtOAc andacidified with 2M KHSO4 (pH about 1). The acidic water phase wasextracted with 4×75 ml of EtOAc and the combined organic phase waswashed with 1×40 ml of H₂ O, 1×40 ml of brine and dried (MgSO₄).Evaporation of the solvent gave 2.0 g (78%) of pure product as a whitesolid.

1H-NMR (CDCl₃, 500 MHz, mixture of two rotamers): δ1.4 and 1.5 (2 s,9H), 2.0-2.1 (m, 1H), 2.3-2.4 (m, 1H), 3.45-3.88 (m, 3H), 4.3 and 4.45(2d, 1H), 7.2-7.4 (m, 5H).

Boc-(R,S)Pro(3-Ph)-OH

Prepared as above starting from a cis/trans mixture ofH-(R,S)Pro(3-Ph)-OH.

Boc-(R)Dph-OH

Prepared according to the method described by K. Hsich et.al. in J. Med.Chem., 32, p. 898 (1989) from H-(R)Dph-OH.

Boc-(R)Hop-OH

Prepared by the same procedure as described for Boc-(R)Cha-OH startingfrom H-(R)Hop-OH.

¹ H-NMR (300 MHz, CDCl₃): δ1.45 (s, 9H), 2.00 (m, 1H), 2.22 (m, 1H),2.75 (bt, 2H), 4.36 (bs, 1H), 5.05 (bs, 1H), 7.15-7.33 (m, 5H).

Deprotection Procedures

(a) The protected peptide was dissolved in EtOH (95%) and hydrogenatedover 5% Pd/C at atmospheric pressure in the presence of an excess of TFAor HOAc (>2 eq.) for about 1-4 h. The catalyst was filtered off, thesolvent evaporated and the final peptide (TFA or HOAc salt) was isolatedas a white powder after freeze drying (H₂ O).

(b) The same as in (a) except that EtOH/H₂ O (ca:5/1) was used assolvent.

(c) The same procedure as in (a) but MeOH was used as solvent.

(d) The same procedure as in (a) but 2M HCl was used as acid to give theHCl-salt.

(e) Hydrolysis of esters, an illustrative example:

EtOOC-CH₂ -(R)Cha-Pro-Nag×2 HOAc (0.4 mmol) was dissolved in 1.5 ml ofMeOH and 1.2 ml (1.2 mmol) of 1M NaOH was added at room temperature.After 3 h the methanol was evaporated and an excess HOAc was added tothe residue and the mixture was freeze dried and purified by RPLC (CH₃CN/0.1M NH₄ OAc (70/30)). The pure product was obtained as a powder in73% yield after freeze drying from water.

(f) Cleavage of t-butyl esters, an illustrative example:

The t-butyl ester was dissolved in an excess of TFA. After stirring for2 h at room temperature the TFA was evaporated. Purification bytreatment with activated charcoal in water-ethanol was followed byfreeze drying from water giving the desired compounds.

Preparation of Starting Materials

H-pic-OEt×HCl

L-Pipecolinic acid, 4.0 g (0.031 mol), was slurried in 100 ml of abs.ethanol and HCl (g) was briefly bubbled through until a clear solutionwas obtained. It was cooled in an ice bath and 17 ml of thionyl chloridewas added dropwise over 15 min. The ice bath was removed and the mixturewas refluxed for 2.5 h. The solvent was evaporated and the product wasobtained as its hydrochloride salt in a quantitative yield.

¹ H-NMR (300 MHz, D₂ O): δ1.33 (t, 3H), 1.8-2.1 (m, 5H), 2.3-2.5 (m,1H), 3.1-3.3 (m, 1H), 3.5-3.7 (m, 1H), 4.14 (dd, 1H), 4.44 (q, 2H).

H-Pic-OMe×HCl

Prepared in the same way as described for H-Pic-OEt×HCl by replacingEtOH with MeOH.

H-Aze-OEt×HCl

Prepared in the same way as described for H-Pic-OEt×HCl from H-Aze-OH.

H-Pic(4-(S)Me)-OEt×HCl

Prepared in the same way as described for H-Pic-OEt×HCL fromH-Pic(4-(S)Me)-OH (purchased from Synthelec, Lund, Sweden).

H-(R)Pic(4-(R)Me)-OEt×HCl

Prepared in the same way as described for H-Pic-OEt×HCl fromH-(R)Pic(4-(R)Me)OH (purchased from Synthelec, Lund, Sweden).

H-(R)Dph-OH

Prepared by the general method given by A. Evans et. al. in JACS, 112,4011 (1990).

H-(R,S)Pic(4,5-dehydro)-oEt

H-(R,S)Pic(4,5-dehydro)-OH, 3.05 g (18.1 mmol) (Prepared according tothe procedure by Burgstahler et. al. J. Org. Chem, 25, 4, p. 489-92(1960), was dissolved in 75 ml EtOH/HCl (saturated) and the mixture wasrefluxed for 5 hours. The solvent was evaporated and the remainingresidue was dissolved in water, made alkaline with sodium hydroxide (aq)and extracted three times with ethylacetate. Drying (Na₂ SO₄) and carefull evaporation gave 2,05 g (71%) of the title compound.

¹ H-NMR (CDCl₃): δ1.28 (t, 3H), 1.88 (bs, NH) 2.2-2.4 (m, 2H), 3.45 (bs,2H), 3.57 (dd, 1H), 4.21 (q, 2H), 5.68-5.82 (m, 2H).

Boc-(R)Cgl-OH

Boc-(R)Pgl-OH was hydrogenated over 5% Rh/Al₂ O₃ in MeOH at 5 Mpa.Filtration and evaporation of the solvent gave the title compound whichwas used without further purification.

¹ H-NMR (300 MHz,CDCl₃): δ0.9-1.7 (m, 20H), 4.0-4.2 (m, 1H), 5.2 (d,1H).

Boc-(R)Dch-OH

Boc-(R)Dph-OH, 0.75 g (2.2 mmol), was dissolved in 25 ml of MeOH and acatalytic amount of 5% Rh/Al₂ O₃ was added. The mixture was hydrogenatedat 5 Mpa, 50° C. for 40 h, filtered and evaporated to give 0.72 g (93%)of the title compound.

¹ H-NMR (CDCl₃): δ0.9-2.0 (m, 32H), thereof 1.45 (bs, 9H), 4.55 (bd) and4.9 (bd); two rotamers integrating for a total of 1H, 5.7-6.1 (broad,NH).

H-(R)Pro(5-(S)Me)-OMe

Prepared according to the procedure given by B. Gopalan et.al. in J.Org. Chem., 51, 2405, (1986).

H-Mor-OH

Prepared according to the method of K. Nakajima. et al. Bull. Chem. Soc.Jpn., 51 (5), 1577-78, 1978 and ibid 60, 2963-2965, 1987.

H-Mor-OEt×HCl

Prepared in the same way as H-Pic-OEt×HCl from H-Mor-OH.

Boc-(R)Cha-OSu

Boc-(R)Cha-OH (1 eq.), HOSu (1.1 eq) and DCC or CME-CDI (1.1 eq) weredissolved in acetonitrile (about 2.5 ml/mmol acid) and stirred at roomtemperature over night. The precipitate formed during the reaction wasfiltered off, the solvent evaporated and the product dried in vacuo.(When CME-CDI was used in the reaction the residue, after evaporation ofthe CH₃ CN, was dissolved in EtOAc and the organic phase washed withwater and dried. Evaporation of the solvent gave the title compound).

¹ H-NMR (500 MHz, CDCl₃, 2 rotamers ca: 1:1 ratio) δ0.85-1.1 (m, 2H),1.1-1.48 (m, 4H), 1.5-1.98 (m, 16H; thereof 1.55 (bs, 9H)), 2.82 (bs,4H), 4.72 (bs, 1H, major rotamer), 4.85 (bs, 1H, minor).

Boc-(Me)(R)Cha-OSu

(i) Boc-(Me)(R)Cha-OH

A solution of 11,9 g (42.6 mmol) Boc-(Me)(R)Phe-OH in 150 ml MeOH washydrogenated over 5% Rh/Al₂ O₃ at 0,28 Mpa for 24 h. Filtration of thecatalyst and evaporation of the solvent gave the product as a whitesolid (95% yield) which was used in the next step without furtherpurification.

¹ H-NMR (500 MHz, CDCl₃, mixture of two rotamers ca: 1/1). δ0.8-1.1 (m,2H), 1.1-1.9 (m, 20H, thereof 1.47 and 1.45 (s, 9H)), 2.82 and 2.79 (s,total 3H), 4.88 and 4.67 (m, total 1H).

(ii) Boc-(Me)(R)Cha-OSu

Prepared in the same way as described for Boc-(R)Cha-OSu- fromBoc-(Me)(R)Cha-OH.

Boc-(R)Cha-Pro-OSu

(i) Boc-(R)Cha-Pro-OH

H-(S)Pro-OH (680 mmol) was dissolved in 0.87M sodium hydroxide (750 ml).Boc-(R)Cha-OSu (170 mmol) dissolved in DMF (375 ml) was added dropwiseduring 20 min. The reaction mixture was stirred at room temperature for20 h. The mixture was acidified (2M KHSO₄) and extracted three timeswith ethyl acetate. The organic layers were combined and washed threetimes with water and once with brine. After drying over sodium sulphateand evaporation of the solvent, the syrupy oil was dissolved in diethylether, the solvent evaporated and finally the product dried in vacuo toyield Boc-(R)Cha-Pro-OH as a white powder in almost quantitative yield.

¹ H-NMR (500 MHz, CDCl₃, minor rotamer 10%) δ0.8-1.05 (m, 2H), 1.05-1-55(m, 15H; thereof 1.5 (bs, 9H)), 1.55-1.8 (m, 5H), 1.8-2.15 (m, 3H), 2.47(m, 1H), 3.48 (m, 1H), 3.89 (m, 1H), 4.55 (m, 2H), 5.06 (m, 1H); minorrotamer signals 2.27 (m, 1H), 3.58 (m, 1H), 4.33 (m, 1H), 5.0 (m, 1H)

(ii) Boc-(R)Cha-Pro-OSu

Prepared in the same way as described for Boc-(R)Cha-OSu- fromBoc-(R)Cha-Pro-OH.

¹ H-NMR (500 MHz, CDCl₃, 2 rotamers, 5:1 ratio) δ0.78-1.05 (m, 2H),1.05-1.83 (m, 20H; thereof 1.43 (bs, 9H)), 1.83-2.26 (m, 3H), 2.32 (m,1H), 2.72-2.9 (m, 4H), 3.2 (m, 1H, minor rotamer), 3.52 (m, 1H, major),3.68 (m, 1H, minor rotamer), 3.89 (m, 1H, major), 4.31 (bq, 1H, minorrotamer), 4.56 (bq, 1H, major), 4.71 (bt, 1H, major rotamer), 4.93 (bt,1H, minor), 5.22 (bd, 1H, major rotamer), 5.44 (bd, 1H, minor).

Z-(R)Cha-Pro-OSu

Prepared in the same way as Boc-(R)Cha-Pro-OSu from Z-(R)Cha-OH.

Boc-(R)Cha-Pic-OSu

(i) Boc-(R)Cha-Pic-OEt

Boc-(R)Cha-OH, 6.3 g (0.023 mol), was dissolved in 150 ml of CH₂ Cl₂.The solution was cooled in an ice bath and 6.3 g (0.047 mol) ofN-hydroxybenzotriazole and 11.2 g (0.0265 mol) of CME-CDI were added.The ice bath was removed after 15 min and the reaction mixture wasstirred for 4 h at room temperature. The solvent was evaporated and theresidue dissolved in 150 ml of DMF and cooled in an ice bath.H-Pic-OEt×HCl, 4.1 g (0.021 mol) was added and the pH adjusted toapproximately 9 by addition of N-methylmorpholine. The ice bath wasremoved after 15 min and the reaction mixture was stirred for 3 days.The solvent was evaporated and the residue was dissolved in ethylacetate and washed with dilute KHSO₄ (aq), NaHCO₃ (aq) and water. Theorganic layer was dried (Na₂ SO₄) and evaporated to give 7.7 g (89%) ofBoc-(R)Cha-Pic-OEt which was used without further purification.

¹ H-NMR (500 MHz, CDCl₃, 2 rotamers, 3:1 ratio ) δ0.7-1.0 (m, 2H),1.1-1.9 (m, 29H; thereof 1.28 (t, 3H)), 1.45 (bs, 9H), 2.01 (bd, 1H,major rotamer), 2.31 (bd, 1H), 2.88 (bt, 1H, minor), 3.30 (bt, 1H,major), 3.80 (bd, 1H, major), 4.15-4.3 (m, 2H), 4.5-4.7 (m, 2H, minor),4.77 (bq, 1H, major), 4.90 (bd, 1H, minor), 5.28 (bd, 1H, major), 5.33(bd,1H, major).

(ii) Boc-(R)Cha-Pic-OH

Boc-(R)Cha-Pic-OEt, 5.6 g (0.014 mol), was mixed with 100 ml of THF, 100ml of water and 7 g of LiOH. The mixture was stirred at room temperatureovernight. The THF was evaporated and the aqueous solution was acidifiedwith KHSO₄ (aq) and extracted three times with ethyl acetate. Thecombined organic phase was washed with water, dried (Na₂ SO₄) andevaporated to give 4.9 g (94%) of Boc-(R)Cha-Pic-OH which was usedwithout further purification. The compound can be crystallized fromdiisopropyl ether/hexane.

¹ H-NMR (500 MHz, CDCl₃, 2 rotamers, 3.5:1 ratio) δ0.8-1.1 (m, 2H),1.1-2.1 (m, 27H; thereof 1.43 (s, 9H, major rotamer), 1.46 (s, 9H,minor)), 2.33 (bd, 1H), 2.80 (bt, 1H, minor), 3.33 (bt, 1H, major), 3.85(bd, 1H, major), 4.57 (bd, 1H, minor), 4.68 (m, 1H, minor), 4.77 (bq,1H, major), 5.03 (bs, 1H, minor), 5.33 (bd, 1H, major), 5.56 (m, 1H,major).

(iii) Boc-(R)Cha-Pic-OSu

Boc-(R)Cha-Pic-OH (1 g, 2.6 mmol) was dissolved in DMF (15 ml) at roomtemperature and then cooled to -18° C., a temperature which wasmaintained during the additions of the reactants. Hydroxy succinimid(0.60 g, 5.2 mmol) was added and the reaction mixture was stirred for afew minutes until the crystals were dissolved. Dicyclohexyl carbodiimid(0.56 g, 2.7 mmol) dissolved in DMF (10 ml) and precooled was addeddropwise to the reaction mixture. After a few minutes at -18° C. thereaction mixture was put into a water bath at 20° C. for 2 h understirring. The solvent was evaporated, ethyl acetate (40 ml) was addedand the precipitated urea was filtered off.

The organic phase was washed once with water, twice with 0.3M KHSO₄,twice with diluted NaHCO₃, once with water, once with brine and dried(Na₂ SO₄). The solvent was evaporated and the product dried in vacuo toyield 1.16 g (93%) of the product. According to ¹ H-NMR the productcontained two diastereoisomers (epimers in Pic, S/R) in a ratio of 95/5.

¹ H-NMR (300 MHz, CDCl₃, major diastereomer) δ0.7-2.0 (m, 27H; thereof1.46 (bs, 9H)), 2.29 (bd, 1H), 2.85 (bs, 4H), 3.40 (m, 1H), 4.5-4.8 (m,1H), 5.1-5.4 (m, 1H), 5.70 (bd, 1H, major).

Boc-(R)Cha-Mor-OSu

Prepared in the same way as Boc-(R)Cha-Pic-OSu from H-Mor-OEt×HCl exceptthat CH₃ CN was used as solvent instead of DMF in the formation of theOSu-ester.

Boc-(Me)(R)Cha-Pro-OSu

Prepared in the same way as Boc-(R)Cha-Pro-OSu from Boc-(Me)-(R)Cha-OH.

Boc-(Me)(R)Cha-Pic-OSu

Prepared in the same way as Boc-(R)Cha-Pic-OSu from Boc-(Me)(R)Cha-OH.

Boc-(R,S)Pro(3-Ph)-Pro-OSu

Prepared in the same way as Boc-(R)Cha-Pro-OSu fromBoc-(R,S)Pro(3-Ph)-OH.

Boc-(R,S)Pro(3-(trans)Ph)-Pro-OSu

(i) Boc-(R,S)Pro(3-(trans)Ph)-Pro-OBn

To a slurry of 1.0 g of Boc-(R,S)Pro(3-(trans)Ph)-OH (3.43 mmol, 1 eq.),1.04 g of H-Pro-OBn×HCl (4.29 mmol, 1.25 eq.), 0.04 g of HOBt (0.24mmol, 0.07 eq.) in 15 ml DMF was added 1.83 g of CME-CDI (4.29 mmol,1.25 eq.) and 0.525 ml of NMM (4.73 mmol, 1.38 eq.) at room temperature.After stirring an additional 4 days the solvent was evaporated and theresidue taken up in 200 ml EtOAc. The organic phase was washed with 2×40ml of H₂ O, 2×25 ml of 1M KHSO₄, 2×25 ml of 1M NaOH, 2×25 ml of H₂ O anddried (MgSO₄). Evaporation of the solvent and flash chromathography (CH₂Cl₂ /MeOH, 97/3) gave the pure product (44% yield) as a ca: 1:1 mixtureof diastereomers.

(ii) Boc-(R,S)Pro(3-(trans)Ph)-Pro-OH

The benzyl ester from the previous step was removed by hydrogenationover 5% Pd/C in EtOH at atmospheric pressure for 4 h. Filtration andevaporation gave the pure product as a ca: 1:1 mixture of diastereomersin quantitative yield.

¹ H-NMR (CDCl₃, 500 MHz, two diastereomers each consisting of tworotamers): δ1.3-2.4 (m+4 s from the Boc groups, total 14H), 2.5-2.9 (m,total 1H), 3.2-3.9 (m, total 5H), 4.3-4.65 (m, total 2H), 7.2-7.5 (m,5H).

(iii) Boc-(R,S)Pro(3-(trans)Ph)-Pro-OSu

Prepared according to the procedure described for Boc-(R)Cha-OSu fromBoc-(R,S)Pro(3-(trans)Ph)-Pro-OH.

Boc-(R,S)Pro(3-(trans)Ch)-Pro-OSu

(i) Boc-(R,S)Pro(3-(trans)Ch)-Pro-OH

Boc-(R,S)Pro(3-(trans)Ph)-Pro-OH was hydrogenated over 5% Rh/Al₂ O₃ inmethanol together with a stall amount of HOAc for 7 days at 0,34 Mpa.Filtration of the catalyst, evaporation of the solvent and flashchromatograpy (CH₂ Cl₂ /MeOH, 94/6) gave the pure product as a whitesolid (mixture or two diastereomers).

(ii) Boc-(R,S)Pro(3-(trans)Ch)-Pro-OSu

Prepared according to the procedure described for Boc-(R)Cha-OSu fromBoc-(R,S)Pro(3-(trans)Ch)-Pro-OH.

Boc-(R)Hoc-Pro-OH

(i) Boc-(R)Hoc-OH

Boc-(R)Hop-OH, 3.2 g (11.46 mmol) was dissolved in methanol (75 ml).Rhodium on activated aluminium oxide (Rh/Al₂ O₃), 0,5 g was added andthe mixture stirred in hydrogen atmosphere at 0.41 MPa for 18 h. Thecatalyst was filtered off through celite and the solvent evaporatedgiving the product in almost quantitative yield.

¹ H-NMR (500 MHz, CDCl₃): δ0.90 (m, 2H), 1.08-1.33 (m, 6H), 1.43 (s,9H), 1.60-1.74 (m, 6H), 1.88 (bs, 1H), 4.27 (bs, 1H).

(ii) Boc-(R)Hoc-OSu

Prepared in the same way as described for Boc-(R)Cha-OSu fromBoc-(R)Hoc-OH.

(iii) Boc-(R)Hoc-Pro-OH

Prepared in the same way as described for Boc-(R)Cha-Pro-OH fromBoc-(R)Hoc-OSu.

¹ H-NMR (500 MHz, CDCl₃): δ0.80-0.94 (m, 2H), 1.05-1.36 (m, 7H),1.36-1.48 (bs, 9H), 1.48-1.78 (m, 7H), 1.98-2.14 (m, 2H), 2.34 (m, 1H),3.48 (m, 1H), 3.85 (m, 1H), 4.43 (m, 1H), 4.52 (bd, 1H), 5.26 (bd, 1H),signals of a minor rotamer appears at: δ1.92, 2.25, 3.58, 4.20 and 4.93.

Boc-(R)Hoc-Pic-OH

(i) Boc-(R)Hoc-Pic-OMe

Prepared the same way as described for Boc-(R)Cha-Pic-OEt fromBoc-(R)Hoc-OH and H-Pic-OMe×HCl.

(ii) Boc-(R)Hoc-Pic-OH

Prepared in the same way as described for Boc-(R)Cha-Pic-OH fromBoc-(R)Hoc-Pic-OMe.

¹ H-NMR (500 MHz, CDCl₃): δ0.82-0.97 (m, 2H), 1.10-1.36 (m, 7H),1.36-1.50 (bs, 9H), 1.50-1.82 (m, 11H), 2.35 (bd, 1H) 3.28 (bt. 1H),3.85 (bd, 1H) 4,63 (m, 1H), 5.33 (bs, 1H), 5.44 (bd, 1H), signals of aminor rotameter appears at: δ1.88, 2.80, 4.25, 4.55 and 4.97.

Boc-(R)Cha-Aze-OH

Prepared in the same way as described for Boc-(R)Cha-Pic-OH fromH-Aze-OEt×HCL.

Boc-(R)Cha-Pic(4-(S)Me)-OH

Prepared in the same way as described for Boc-(R)Cha-Pic-OH fromH-Pic(4-(S)Me)-OEt×HCl except that CH₂ Cl₂ was used as solvent.

Boc-(R)Cha-(R)Pic(4-(R)Me)-OSu

(i) Boc-(R)Cha-(R)Pic(4-(R)Me)-OEt

Prepared in the same way as described for Boc-(R)Cha-Pic-OEt fromH-(R)Pic(4-(R)Me)-OEt×HCl.

(ii) Boc-(R)Cha-(R)Pic(4-(R)Me)-OH

Prepared by using the deprotection (e) on the product (i) above.

(iii) Boc-(R)Cha-(R)Pic(4-(R)Me)OSu

Prepared in the same way as described for Boc-(R)Cha-Pic-OSu fromBoc-(R)Cha-(R)Pic(4-(R)Me)-OH.

Boc-(R)Cha-(R,S)Pic(4,5-dehydro)-OH

Prepared according to the procedure described for Boc-(R)Cha-Pic-OH fromH-(R,S)Pic(4,5-dehydro)-OEt.

Boc-(R)Cgl-Pic-OH

(i) Boc-(R)Cgl-Pic-OMe

Pivaloyl chloride (1.000 mL, 8.1 mmol) was added to a solution ofBoc-(R)Cgl-OH (2.086 g, 8.1 mmol) and triethyl amine (1.13 mL, 8.1 mmol)in toluene (25 mL) and DMF (5 mL). A mixture of H-Pic-OMe×HCl (1.46 g,8.1 mmol) and triethyl amine (1.13 mL, 8.1 mmol) in DMF (20 mL) wassubsequently added at ice bath temperature. The reaction mixture wasslowly allowed to warm up to room temperature and after 24 h it wasdiluted with water and extracted with toulene. After washing with 0.3MKHSO₄, 10% Na₂ CO₃ and brine the solvent was removed in vacuo to give2.52 g (81%) of colorless oil which was used without furtherpurification.

¹ H-NMR (500 MHz, CDCl₃, 2 rotamers, 5:1 ratio) δ0.8-1.8 (m, 25H), 2.25(d, 1H), 2.75 (t, 1H, minor rotamer), 3.3 (t, 1H), 3.7 (s, 3H), 3.85 (d,1H), 4.3 (t, 1H, mincr rotamer), 4.5-4.6 (m, 1H), 5.25 (d, 1H), 5.30 (d,1H).

(ii) Boc-(R)Cgl-Pic-OH

Prepared according to the procedure for hydrolysis of Boc-(R)Cha-Pic-OEtusing the product from (i) above. The product was crystallized fromdi-isopropyl ether and hexane.

¹ H-NMR (500 MHz, CDCl₃, 2 rotamers, 5:1 ratio) δ0.8-1.8 (m, 25H), 2.3(d, 1H), 2.8 (t, 1H, minor rotamer), 3.3 (t, 1H), 3.9 (d, 1H), 4.4 (t,1H, minor), 4.5-4.6 (m, 1H), 5.1 (s, 1H, minor rotamer), 5.3 (d, 1H),5.40 (d, 1H).

Boc-(R)Dph-Pic-OH

Prepared in the same way as described for Boc-(R)Cha-Pic-OH fromBoc-(R)Dph-OH.

Boc-(R)Dch-Pic-OH

Prepared in the same way as described for Boc-(R)Cha-Pic-OH fromBoc-(R)Dch-OH.

Boc-(R)Cha-Pro(5-(S)Me)-OH

Prepared in the same way as described for Boc-(R)Cha-Pic-OH fromH-Pro(5-(S)Me)-OMe.

Boc-Nag(Z)

(i) N-Bensyloxycarbonyl-O-methyl isourea

To a stirred solution of concentrated aqueous NaOH (2.8 L, 50% w/w,19.1M, 53 mol) and water (32 L) at 18° C. was added in two portionsO-methylisourea hemisulphate (1.7 kg, 94%, 13.0 mol) and 0-methylisoureahydrogensulphate (1.57 kg, 99%, 9.0 mol). The reaction mixture wascooled to 3°-50° C. Benzyl chloroformiate (3.88 kg, 92%, 20.9 mol) wasadded over a 20 minutes period under cooling and vigorous stirring. Thereaction temperature went from 3° to 8° C. during the addition of Z-Cl.The addition funnel was rinsed with 5 liters of water which was added tothe reactor. The reaction mixture was stirred at 0°-3° C. for 18 h,filtered and the crystals was washed with cooled (3° C.) water (10 L).Vacuum drying 25° C., 10-20 mbar) for 48 h gave 3.87 kg (89%) of thetitle compound as a white crystalline powder.

(ii) Boc-Nag(Z)

To a stirred solution Boc-NH-(CH₂)₃ -NH₂ ×HCl (prepared according toMattingly P. G., Synthesis, 367 (1990)) (3.9 kg, 18.5 mol) iniso-propanol (24 kg) at 60°-70° C. was added in portions over a 30minutes period KHCO₃ (4.2 kg, 42 mol). A slow evolution of CO₂ (g)occurs. The mixture was stirred for another 30 minutes followed byaddition in portions over a 30 minutes periodN-bensyloxycarbonyl-O-methyl isourea (3.74 kg, 18.0 mol). The reactionmixture was stirred at 65°-70° C. for 16 h, cooled to 20° C. andfiltered. The precipitate was washed with iso-propanol (10+5 L). Thecombined filtrates was concentrated at reduced pressure keeping theheating mantle not warmer than 65°-70° C. When approximately 45 literswas distilled off EtOAc (90 L) was added. The reaction mixture wascooled to 20°-25° C., washed with water (10 and 5 L) and brine (5 L),and dried with Na₂ SO₄ (2 kg). After stirring the reaction mixture wasfiltered and the filter cake was washed with EtOAc (11 and 7 L). Thecombined filtrates were concentrated at reduced pressure keeping theheating mantle not warmer than 40°-50° C. When approximately 90 litersof EtOAc was distilled off, toluene (25 L) was added and the evaporationcontinued.

After collection of approximately another 18 liters of destillate,toulene (20 L) was added under vigorous stirring and the resultingmixture was cooled to -1° to 0° C. and gently stirred over night (17 h).The crystal slurry was filtered and the product was washed with cooledtoluene (10 and 5 L). Vacuum drying (10-20 mbar, 40° C.) for 24 h gave4.83 kg (13.8 mol, 76%) of Boc-Nag(Z).

¹ H-NMR (300 MHz, CDCl₃): δ1.41 (s, 9H), 1.6-1.7 (m, 2H), 3.0-3.3 (m,4H), 4.8-5.0 (bs, 1H), 5.10 (s, 2H), 7.2-7.4 (m, 5H).

Boc-Agm(Z)

(i) Boc-Agm

To a slurry of 14.95 g (65.5 mmol, 1 eq.) of agmatine sulphate(Aldrich), 13.7 ml of Et₃ N (98.25 mmol, 1.5 eq.), 165 ml of H₂ O and165 ml of THF was added 21.5 g (98.25 mmol, 1.5 eq.) of (Boc)₂ O during5 minutes at room temperature. The mixture was stirred vigorously overnight, evaporated to dryness and the residue was washed with 2×100 ml ofEt₂ O to give Boc-Agm as a white powder which was used without furtherpurification in the next step.

(ii) Boc-Agm(Z)

To a cold (+5° C.) slurry of the crude Boc-Agm from the previous step(ca: 65.5 mmol) in 180 ml of 4N NaOH and 165 ml of THF was added 24 ml(169 mmol, 2.5 eq) of benzyl chloroformate during 10 minutes. Afterstirring at room temperature for 4 h methanol (150 ml) was added and thestirring was continued for an additional 20 h at room temperature. Theorganic solvent was evaporated and 200 ml of H₂ O was added to theresidue. The basic water phase was extracted with 1×300 ml and 2×200 mlof EtOAc. The combined organic phases was washed with H₂ O (2×100 ml),brine (1×100 ml) and dried (MgSO₄). Evaporation of the solvent and flashchromathography (CH₂ Cl₂ /MeOH, a stepwise gradient of 97/3, 95/5 and9/1 was used) gave 14.63 g (58%) of pure Boc-Agm(Z) as a white powder.

¹ H-NMR (CDCl₃, 500 MHz): δ1.35-1.40 (m, 2H), 1.45 (s, 9H), 1.5-1.6 (m,2H), 3.0-3.2 (m, 4H), 4.65 (bs, 1H), 5.1 (s, 2H) 7.25-7.40 (m, 5H).

¹³ C-NMR (CDCl₃, 75.5 MHz) δ25.44, 27.36, 28.21, 65.83, 79.15, 127.47,127.66, 128.14, 137.29, 156.47, 161.48, 163.30.

Boc-NH-(CH₂)₃ -N₃

Prepared according to the method described by Mattingly P. G., inSynthesis 1990, 367.

Z--NH--(CH₂)₂ --NH₂

To a cold solution of 6 g ethylene diamine (0.1 mol) and 22 ml triethylamine in 20 ml of chloroform was added 2.5 g of Z-OSu dissolved in 5 mlof chloroform. The mixture was allowed to reach room temperature andleft over night under stirring. Filtration, evaporation of the solventand flash chromatography (CH₂ Cl₂ /MeOH(NH₃ -saturated), 95/5) gave 0.9g (46%) of the title compound.

¹ H-NMR (300 MHz, CDCl₃): δ1.27 (s, 2H), 2.85 (t, 2H), 3.24 (q, 2H),5.14 (s, 2H), 7.22-7.40 (m, 5H).

Agm×HCl

Prepared from Agm×H₂ SO₄ (Aldrich) by exchanging the hydrogen sulphateion for chloride on an ion exchange column.

H-Nag(Z)×2 HCl

Prepared by bubbling HCl(e) into a solution of Boc-Nag(Z) in EtOAcfollowed by evaporation of the solvent.

BnOOC--CH₂ --NH--CO--CH₂ --Br

To a solution of p-TsOH×H-Gly-OBn (5 mmol) and triethyl amine (5 mmol)in 10 ml of CH₂ Cl₂ was added 2-bromoacetic acid (5 mmol) dissolved in10 ml of CH₂ Cl₂ and dicyclohexyl carbodiimide (5 mmol). The mixture wasstirred at room temperature over night and filtered. The organic phasewas washed twice with 0.2M KHSO₄, 0.2M NaOH, brine and dried.Evaporation and flash chromatography (CH₂ Cl₂ /MeOH, 95/5) gave aquantitative yield of the desired compound.

¹ H-NMR (300 MHz, CDCl₃): δ=3.89 (s, 2H), 4.05-4.11 (d, 2H), 5.19 (s,2H), 7.06 (bs, 1H), 7.3-7.4 (m, 5H)

BnOOC--CH₂ --OCO--CH₂ --Br

A mixture of 2.8 g (0.020 mmol) bromoacetic acid, 4.2 g (0.020 mmol) ofbenzyl bromoacetate and 2.0 g (0.020 mmol) of triethylamine in 25 ml ofEtOAc was refluxed for 3 h. It was diluted with more EtOAc and cooled.The solution was washed with dilute HCl and thereafter with NaHCO₃ (aq)and finally with water. Drying (Na₂ SO₄) and evaporation followed byflash chromatography (heptane/etylacetate, 75/25) gave the titlecompound in 26% yield.

¹ H-NMR (500 MHz, CDCl₃): δ3.95 (s, 2H), 4.75 (s, 2H), 5.23 (s, 2H),7.35-7.45 (m, 5H).

BnO-(CH₂)₃ -OTf

Propanediol monobenzyl ether (0.83 g, 5 mmol) was dissolved in drypyridine (0.6 g, 7 mmol) and dichloromethane (20 ml) and cooled to -15°C. Triflic anhydride, precooled to -15° C., was added and the reactionmixture stirred for 45 min under which the temperature was allowed torize to 15° C. The solvent was evaporated and the product dissolved inhexane/ethyl-acetate 4:1 (10 ml) and filtered through silica.

Finally the solvent was evaporated and the product dried in vacuo toyield 0.95 g (64%) of 1-benzyloxy 3-trifluoromethanesulfonylpropanewhich was used directly (see Example 21).

¹ H-NMR (500 MHz, CDCl₃): δ2.12 (m, 2H), 3.6 (t, 2H), 4.51 (s, 2H), 4.72(t,2H), 7.22-7.42 (m, 5H).

BnO--(CH₂)₂ --CHO

Prepared by Swern oxidation (described by D. Swern et al., J. Org.Chem., 1978, 2480-82) of BnO--(CH₂)₃ --OH.

¹ H-NMR (300 MHz, CDCl₃): δ2.63 (dt, 2H), 3.80 (t, 2H), 4.51 (s, 2H),7.30 (m, 5H), 9.76 (bt, 1H).

Br--(S)CH(CH₂ OBn)--COOBn

(i) Br--(S)CH(CH₂ OBn)--COOH

O-Benzylserine (3.9 g, 19 mmol) in water (10 ml) was added to a solutionof sodium bromide (11 g, 107 mmol) in water (20 ml) and sulphuric acid(2 g, 20 mmol). The reaction mixture was cooled to -10° C. and NaNO₂(1.73 g, 25 mmol) was added under vigorous stirring. Another portion ofwater was added to the thick mixture followed, after a few minutes, byH₂ SO₄ (1 g, 10 mmol). The mixture was stirred at ambient temperatureover night after which it was extracted twice with EtOAc (100 ml). Thecombined organic phase was washed twice with water and once with brineand dried (Na₂ SO₄). Evaporation of the solvent gave 3.7 g (75%) of thetitle compound as a yellow oil which was pure enough to use directly inthe next step.

(ii)Br--(S)CH(CH₂ OBn)--COOBn

To a solution of the crude product from (i) above (2.6 g, 10 mol) in drybenzene (25 ml) was added oxalyl chloride (2.6 g, 20.5 nmol) andmolecular sieves (4 Å, 1 g). The mixture as stirred at ambienttemperature under an atmosphere of Argon for 18 h. The molecular sieveswas removed by filtration and the solvent evaporated. The slightlyyellow residue was dissolved in CH₃ CN (10 ml) and benzyl alcohol (1 g,9.2 mmol) was added. The mixture was stirred at ambient temperature for5 h. The solvent was evaporated and the residue dissolved in Et₂ O andwashed once with 1M NaOH, water, brine and dried (Na₂ SO₄) Evaporationof the solvent followed by flash chromatography (CH₂ Cl₂ /MeOH, 95/5)gave 1.8 g (67%) of the desired compound.

¹ H-NMR (500 MHz, CDCl₃): δ3.82 (dd, 1H), 3.99 (dd, 1H), 4.38 (dd, 1H),4.56 (s, 2H), 5.23 (s, 2H), 7.23-7.46 (m, 5H).

WORKING EXAMPLES Example 1 H-(R)Cha-Pro-Agm×2 HOAc

(i) Boc-(R)Cha-Pro-Agm×HOAc

Boc-(R)Cha-Pro-OSu (1.7 mmol) and agmatine dihydrochloride (2.0 mmol,1.18 eq) was dissolved in DMF/H₂ O 95:5 (35 ml). Triethylamine was addedto adjust the pH to about 10 and the solution was stirred at roomtemperature for 2 days. The solution was evaporated (5 mm Hg/ 60° C.)until dryness and the crude product was purified by RPLC (CH₃ CN/NH₄ OAc(0.1M), 38:62). The desired compound was obtained as a white powderafter freeze-drying.

¹ H NMR (500 MHz, CDCl₃ /DMSO-d₆ 5:2, Two rotamers, 9:1 δ (majorrotamer): 0.75-0.90 (m, 2H), 1.1-2.05 (m, 19H), 1.35 (s, 9H) 2.98-3.14(m, 4H), 3.37 (q,1H), 3.76 (m, 1H), 4.20 (m,1H), 4.33 (dd, 1H), 6.30 (d,1H), 7.05-7.80 (broad m, 5H), 8.67 (broad d, 1H).

Exchange broadened signals of the minor rotamer are unambiguouslyobserved at δ3.44 (m, 1H), 3.62 (m, 1H), 4.10 (m, 1H), 4.64 (m, 1H),5.56 (d, 1H), 9.08 (m, 1H)

(ii) H-(R)Cha-Pro-Agm×2 HOAc

A solution of Boc-(R)Cha-Pro-Agm (0.2 mmol) in TFA (2 ml) was stirred atroom temperature for 4.5 h. The solvent was evaporated and the remainingoil was subjected to RPLC (CH₃ CN/NH₄ OAc (0.1M), 25:75). The diacetatesalt was obtained as a white powder after repeated freeze-drying.

¹ H NMR (500.13 MHz, D₂ O): δ0.80-0.95 (m, 2H), 1.00-1.21 (m, 3H), 1.32(m, 1H), 1.40-1.78 (m,12H), 1.83-2.00 (m, 2H), 1.90 (s, acetate),2.20(m, 1H), 3.06-3.14(m. 4H), 3.50(m, 1H), 3.67(m, 1H), 4.20-4.30(m,2H).

¹³ C NMR (75.6 MHz, D₂ O): guanidine: δ157.4; carbonyl carbons: δ169.9,174.5.

Example 2 Me-(R)Cha-Pro-Agm×2 HOAc

(i) Boc-(Me)(R)Cha-Pro-Agm

To a solution of 479.6 mg (1 mmol, 1 eq.) of Boc-(Me)(R)Cha-Pro-OSu and500 ml of NMM in 16 ml DMF/H₂ O (15/1) was added 166.5 mg (1.2 mmol, 1.2eq.) of Agm×HCl at room temperature. The reaction was stirred anadditional 70 h and the solvent was evaporated to give a crude productas an oil. This was used without purification in the next step.

(ii) Me-(R)Cha-Pro-Agm×2 HOAc

The crude oil from the previous step was dissolved in 10 ml TFA/CH₂ Cl₂(1:4) at room temperature. After stirring for 2 h 25 min the solvent wasevaporated and the crude product was purified with RPLC (CH₃ CN/NH₄OAc(0.1M), 35/65) to give the desired product as a white powder afterfreeze-drying.

¹ H-NMR (500 MHz, D₂ O): δ0.93-1.05 (m, 2H), 1.10-1.29 (m, 3H),1.33-1.43 (m, 1H), 1.50-1.80 (m, 12H), 1.88-2.10 (m, 2H, 1.92 (s,acetate), 2.27-2.36 (m, 1H), 2.68 (s, 3H), 3.15-3.23 (m, 3H), 3.24-3.31(m, 1H), 3.57-3.66 (m, 1H), 3.76-3.83 (m, 1H), 4.28 (t, 1H), 4.39 (dd,1H).

¹³ C-NMR (125.76 MHz, D₂ O): guanidine: δ157.24; carbonyl carbons:δ174.03, 168.24.

Example 3 HO-(CH₂)₃ -(R)Cha-Pro-Agm×2 HCl

(i) Boc-(R)-Cha-Pro-Agm(Z)

Boc-Agm(Z) (1 eq) was dissolved in TFA/CH₂ Cl₂ (1:4, ca: 6 ml/mmol) andstirred at room temperature for ca: 2 h. The solvent was evaporated andthe product dissolved together with Boc-(R)Cha-Pro-OSu (1 eq) in DMF(ca: 1 ml/mmol), the pH was adjusted with NMM to ca: 9 and the mixturewas stirred at room temperature for 20 h. The solvent was evaporated invacuo, the crude product dissolved in CH₂ Cl₂ and washed three timeswith water and once with brine. After drying (sodium sulphate) thesolvent was evaporated and the product flash chromatographed (CH₂ Cl₂/MeOH) affording Boc-(R)Cha-Pro-Agm(Z) as a white powder.

(ii) H-(R)Cha-Pro-Agm(Z)

Boc-(R)Cha-Pro-Agm(Z) was dissolved in TFA/CH₂ Cl₂ (1:4, ca: 6 ml/mmol)and stirred at room temperature for 2 h. The solvent was evaporated, theproduct dissolved in 0.2M NaOH (20 ml/mmol) and extracted twice withdichloromethane. The organic layers were combined and washed with brine,dried (sodium sulphate) and the solvent evaporated to yieldH-(R)Cha-Pro-Agm(Z) as a white powder.

(iii) BnO-(CH₂)₃ -(R)Cha-Pro-Agm(Z)

H-(R)Cha-Pro-Agm(Z) (1 mmol) was dissolved in methanol (10 ml).Triethylammonium hydrochloride (1 mmol), sodium cyanoborohydride (0.7mmol) and thereafter BnO--(CH₂)₂ --CHO (1.05 mmol) were added and thereaction mixture stirred at room temperature over night. The solvent wasevaporated and the crude product was dissolved in ethyl acetate, washedtwice with water, once with brine and dried over sodium sulphate. Thesolvent was evaporated and the crude product was purified by flashchromatography (EtOAc/MeOH).

(iv) HO-(CH₂)₃ -(R)Cha-Pro-Agm×2 HCl

Prepared by using deprotection procedure (d) on the product (iii) above.

¹ H-NMR (500 MHz, D₂ O): δ0.72 (m, minor rotamer), 0.84 (m, minorrotamer), 0.87-1.03 (m, 2H), 1.03-1-26 (m, 3H), 1.28-1.40 (bs, 1H),1.44-1.80 (m, 11H), 1.80-1.95 (bs, 3H), 1.95-2.10 (bs, 2H), 2.28 (m,1H), 3.04 (m, 1H), 3.08-3.27 (m, 5H), 3.58 (bs, 1H), 3.67 (bs, 2H), 3.78(m, 1H), 4.12 (bd, minor rotamer), 4.30 (m,1H), 4.37 (m, 1H).

¹³ C-NMR (125 MHz, D₂ O): guanidine: δ157.26; carbonyl carbons: δ174.06,168.36.

Example 4 HOOC-CH₂ -(R)Cha-Pro-Agm×HOAc

General Procedure for the alkylation of the N-terminal.

This procedure is described in more general terms and will be referredto in the Examples below together with the alkylating agent used in eachspecific Example.

The peptide to be alkylated (1 eq) and the alkylating agent (1.1-1.2 eq)were dissolved in acetonitrile (ca 10 ml/mmol). Potassium carbonate(2.0-2.2 eq) was added and the reaction mixture stirred at 50°-60° C.until the starting material was consumed (TLC, usually 1-5 h).Filtration, evaporation of the solvent and flash chromatography (CH₂ Cl₂/MeOH, CH₂ Cl₂ /MeOH(NH₃ -saturated) or EtOAc/MeOH, ca 9/1) gave thealkylated product after evaporation of the solvent.

(i) BnOOC-CH₂ -(R)Cha-Pro-Agm(Z)

Prepared from H-(R)Cha-Pro-Agm(Z) (See Example 3) and Br--CH₂ COOBnaccording to the procedure described above.

(ii) HOOC-CH₂ -(R)Cha-Pro-Agm×HOAc

Prepared by using the deprotection procedure (b) on the product (i)above.

¹ H-NMR (300 MHz, MeOD): δ0.9-1.1 (m, 2H), 1.1-2.3 (m, 19H) 1.95 (s,acetate), 3.1-3.2 (m, 4H), 3.2-3.65 (m, 3H), 3.85 (m, 1H), 4.0 (bt, 1H),4.35 (dd, 1H).

¹³ C-NMR (75 MHz, D₂ O): guanidine: δ157.55; carbonyl carbons: δ168.71,171.37 and 174.3.

Example 5 ^(i) Pr-OOC-CH₂ -(R)Cha-Pro-Agm×HOAc

Alkylation as in Example 4 using H-(R)Cha-Pro-Agm(Z) (See Example 3) andBr--CH₂ COO^(i) Pr followed by deprotection procedure (b) gave the titlecompound.

¹ H-NMR (500 MHz, MeOD): δ0.85-1.05 (m, 2H), 1.1-1.35 (m, 9H; thereof1.23 (d, 3H), 1.25 (d, 3H)), 1.35-2.02 (m, 14H) 1.92 (s, acetate), 2.08(m, 1H), 2.2 (m, 1H), 3.07-3.45 (m, 6H), 3.55 (m, 1H), 3.7-3.8 (m, 2H),4.3 (dd, 1H), 5.05 (m, 1H)

¹³ C-NMR (125 MHz, D₂ O): guanidine: δ157.39; carbonyl carbons: δ171.10,172.76 and 174.44.

Example 6 HOOC-CH₂ -(Me)(R)Cha-Pro-Agm×2 TFA

(i) Me-(R)Cha-Pro-Agm(Z)

Prepared from Boc-(Me)(R)Cha-Pro-OSu in the same way as described forH-(R)Cha-Pro-Agm(Z) in Example 3.

(ii) HOOC-CH₂ -(Me)(R)Cha-Pro-Agm×2 TFA

Alkylation as in Example 4 using Me-(R)Cha-Pro-Agm(Z) and Br--CH₂ COOBnfollowed by deprotection procedure (b) gave the title compound.

¹ H-NMR (300 MHz, D₂ O): δ0.9-1.35 (m, 6H), 1.5-2.2 (m, 14H), 2.25-2.45(m, 1H), 3.12 (s, 3H), 3.15-3.35 (m, 4H), 3.6-3.75 (m, 1H), 3.8-3.95 (m,1H), 4.22 (apparent bs, 2H), 4.45 (m, 1H), 4.6 (bt, 1H).

¹³ C-NMR (75.47 MHz, D₂ O): guanidine: δ157.52; carbonyl carbons:δ173.86, 168.79, 167.38.

Example 7 HOOC-(R,S)CH(Me)-(R)Cha-Pro-Agm×HOAc

Alkylation as in Example 4 using H-(R)Cha-Pro-Agm(Z) (See Example 3) andBr--CH(Me)COOBn followed by deprotection procedure (a) gave the titlecompound as a mixture of two diastereomers.

Example 8 HOOC-(RorS)CH(Me)-(R)Cha-Pro-Agm/a×HOAc

Obtained by separating the diastereomers formed in Example 7 using RPLC(CH₃ CN/NH₄ OAc (0.1M), 1/4). This diastereomer came out first of thetwo from the column.

¹ H-NMR (500 MHz, D₂ O; 2 rotamers ca: 5:1 ratio): δ0.74 (m, minorrotamer), 1.01 (m, 2H), 1.10-1.33 (m, 3H), 1.48-1.88 (m, 15H; thereof1.51 (d, 3H)), 1.92-2.12 (m, 3H) 1.96 (s, acetate), 2.30 (m, 1H), 3.20(m, 3H), 3.38 (m, 1H), 3.47 (q, minor rotamer), 3.53-3.68 (m, 2H), 3.73(m, 1H), 4.20 (d, minor rotamer), 4.33 (m, 1H), 4.38 (m, 1H), 4.51 (d,minor rotamer).

¹³ C-NMR (125 MHz, D₂ O): guanidine: δ157.38; carbonyl carbons: δ174.11,173.45, 168.64.

Example 9 HOOC-(RorS)CH(Me)-(R)Cha-Pro-Agm/b×HOAc

The diastereomer that came out after the first one from the column inthe separation in Example 8 is the title compound above.

¹ H-NMR (500 MHz, D₂ O, 2 rotamers ca 9:1 ratio): δ0.88 (m, minorrotamer), 1.05 (m, 2H), 1.12-1.33 (m, 3H), 1.42 (bs, 1H), 1.50-1.88 (m,15H; thereof 1.55 (d, 3H)), 1.93-2.13 (m, 3H) 1.95 (s, acetate), 2.30(m, 1H), 2.40 (m, minor rotamer), 3.22 (t, 2H), 3.28 (t, 2H), 3.64 (m,1H), 3.70 (q, 1H), 3.98 (t, minor rotamer), 4.35 (t, 1H), 4.41 (dd, 1H).

Example 10 HOOC-(RorS)CH(^(n) Pr)-(R)Cha-Pro-Agm/a×HOAc

Alkylation as in Example 4 using H-(R)Cha-Pro-Agm(Z) (See Example 3) andBr--CH(^(n) Pr)COOEt and deprotection procedure (e) followed bydeprotection procedure (b) gave HOOC-(R,S)CH(^(n) Pr)-(R)Cha-Pro-Agm.The title compound was obtained by separating the diastereomers by RPLC(CH₃ CN/NH₄ OAc (0.1M), 1/4) and freeze drying (H₂ O) after evaporationof the solvent. This diastereomer came out first of the two from thecolumn.

¹ H-NMR (300 MHz, MeOD): δ0.8-1.1 (m, 5H; thereof 0.92 (t, 3H)), 1.1-2.1(m, 22H) 1.95 (s, acetate), 2.2 (m, 1H), 3.1-3.35 (m, 5H), 3.48 (m, 1H),3.88 (m, 1H), 4.0 (m, 1H), 4.4 (dd, 1H).

¹³ C-NMR (75 MHz, D₂ O): guanidine: δ157.50; carbonyl carbons: δ168.55and 174.16.

Example 11 HOOC-(RorS)CH(^(n) Pr)-(R)Cha-Pro-Agm/b×HOAc

The other diastereomer from the separation in Example 10 which came outafter the first one from the column is the title compound above.

¹ H-NMR (500 MHz, MeOD): δ0.85-1.05 (m, 5H; thereof 0.95 (t, 3H))1.1-2.08 (m, 22H) 1.9 (s, acetate), 2.14 (m, 1H), 3.1-3.4 (m, 5H), 3.45(m, 1H), 3.62 (m, 1H), 3.80 (m, 1H), 4.34 (dd, 1H).

¹³ C-NMR (75 MHz, D₂ O): guanidine: δ157.53; carbonyl carbons: δ169.01and 174.27.

Example 12 HOOC-(RorS)CH(Ph)-(R)Cha-Pro-Agm/b×HOAc

(i) ^(t) BuOOC-(RorS)CH(Ph)-(R)Cha-Pro-Agm(Z)

A mixture of H-(R)Cha-Pro-Agm(Z) (See Example 3) (0.55 mmol),tert.butyl-(R,S)phenyl bromoacetate (0.66 mmol), K₂ CO₃ (1.4 mmol) inCH₃ CN (10 ml) was stirred at room temperature for 28 h and anadditional 5 h at 60° C. The diastereomeric mixture (ca: 3:1, accordingto NMR) was filtered and evaporated. The remaining oil was twicesubjected to flash chromatography (CH₂ Cl₂ /MeOH, 92/8), which resultedin a complete separation of the two diastereomers (R_(f) =0.36 (minorisomer) and 0.27 (major isomer), respectively).

¹ H NMR of major isomer (500.13 MHz, CDCl₃): δ0.79 (quart,1H), 0.90(quart,1H), 1.06-1.70 (m, H), 1.37 (s,9H), 1.85-2.03 (m,3H), 2.20(m,1H), 3.10-3.24 (m,3H), 3.25-3.38 (m,2H), 3.42 (m,1H), 3.53 (m,1H),4.30 (s,1H), 4.49 (dd,1H), 5.08 (s,2H), 7.19-7.40 (m,10H); broad NHsignals are observed in the region 6.7-8.6.

(ii) HOOC-(RorS)CH(Ph)-(R)Cha-Pro-Agm/b×HOAc

The major isomer (50 mmol) and thioanisole (0.5 mmol) dissolved in TFAwas kept at room temperature for 8 h. After evaporation (0.1 mm Hg) for5 h, the remaining oil was purified on RPLC (CH₃ CN/NH₄ OAc (0.1M), 2:3)to give the title compound after evaporation of the solvent andfreeze-drying.

¹ H NMR (500.13 MHz, MeOD): δ0.85-1.01 (m, 2H), 1.13-1.38 (m, 4H),1.53-2.05 (m, 14H), 1.92 (s, acetate) 2.18 (m, 1H), 3.08-3.26 (m, 3H),3.32-3.45 (m, 2H), 3.64 (m, 1H), 3.93 (t, 1H), 4.37 (dd, 1H), 4.43(s,1H), 7.28-7.50 (m, 5H).

¹³ C NMR (125.6 MHz, MeOD): guanidine: δ158.7; carbonyl carbons: δ173.8,174.7, 177.0.

Example 13 HOOC-(R,S)CH(CH₂ CH₂ Ph)-(R)Cha-Pro-Agm×HOAc

Alkylation as in Example 4 using H-(R)Cha-Pro-Agm(Z) (See Example 3) andBr--CH(CH₂ --CH₂ --Ph)COOEt and deprotection procedure (a) followed bydeprotection procedure (e) gave HOOC-(R,S)CH(CH₂ -CH₂-Ph)-(R)Cha-Pro-Agm.

Example 14 HOOC-(RorS)CH(CH₂ CH₂ Ph)-(R)Cha-Pro-Agm/a×2 TFA

The title compound was obtained by separating the diastereomers obtainedin Example 13 by RPLC (CH₃ CN/NH₄ OAc (0.1M), 2/3) and freeze drying (H₂O TFA) after evaporation of the solvent. This diastereomer came outfirst of the two from the column is the title compound above.

¹ H-NMR (500 MHz, MeOD): δ0.93-1.11 (m, 2H), 1.24 (m, 1H), 1.29-1.40 (m,2H), 1.52-1.85 (m, 11H), 1.89-2.11 (m, 4H), 2.14-2.32 (m, 3H), 2.83 (t,2H), 3.14 (t, 2H), 3.24 (t, 2H), 3.50 (q, 1H), 3.70 (m, 1H), 4.00 (t,1H), 4.36-4.42 (m, 2H), 7.17-7.31 (m, 5H).

¹³ C-NMR (125 MHz, MeOD): guanidine: δ158.66; carbonyl carbons: δ168.08,171.53, 174.16.

Example 15 HOOC-CH₂ -CH₂ -(R)Cha-Pro-Agm×HOAc

(i) BnOOC-CH₂ -CH₂ -(R)Cha-Pro-Agm(Z)

Benzyl acrylate (1.1 eq) and H-(R)Cha-Pro-Agm(Z) (See Example 3) (1 eq)were dissolved in ethanol (20 ml/mmol) and stirred at room temperaturefor 20 h. The solvent was evaporated and the crude product purified byflash chromatography (CH₂ Cl₂ /MeOH(NH₃ -saturated), 95/5). Finally thesolvent was evaporated and the product dried in vacuo.

¹ H-NMR (500 MHz, CDCl₃): δ0.7-0.95 (m, 2H), 1.0-1.5 (m, 10H), 1.5-1.75(m, 5H), 1.75-1.92 (m, 2H), 2.0 (m, 1H), 2.17 (bs, 1H), 2.45 (m, 2H),2.63 (m, 1H), 2.79 (m, 1H), 2.97-3.25 (m, 4H), 3.33 (m, 2H), 3.52 (bt,1H), 4.45 (bd, 1H), 4.95-5.12 (m, 4H), 7.13-7.4 (m, 10H).

(ii) HOOC-CH₂ -CH₂ -(R)Cha-Pro-Agm×HOAc

Prepared by using the deprotection procedure (a) on the product (i)above.

¹ H-NMR (500 MHz, D₂ O): δ0.88 (m, 2H), 1.00-1.23 (m, 3H), 1.33 (bs,1H), 1.42-1.72 (m, 11H), 1.78-2.00 (m, 3H) 1.94 (s, acetate), 2.18 (m,1H), 2.52 (m, 2H), 3.03-3.20 (m, 6H), 3.50 (m, 1H), 3.72 (m, 1H), 4.23(m, 1H), 4.30 (m, 1H).

¹³ C-NMR (125 MHz, D₂ O): guanidine: δ157.25; carbonyl carbons: δ178.07,173.96, 168.24.

Example 16 EtOOC-CO-(R)Cha-Pro-Agm×HOAc

(i) EtOOC-CO-(R)Cha-Pro-Agm(Z)

To a cold (-10° C.) solution of H-(R)Cha-Pro-Agm(Z) (See Example 3)(0.46 g, 0.89 mmol) and NMM (199 mg, 1.97 mmol) in 10 ml of THF wasadded Cl--COCOOEt (134 mg, 0.98 mmol) dissolved in 3 ml of THF. Themixture was kept at -10° C. for one hour after which it was stirred atroom temperature for another hour. The solvent was evaporated and theresidue was dissolved in ethyl acetate. The organic phase was washedtwice with water and dried (Na₂ SO₄). Evaporation of the solvent andcrystallization from EtOAc gave 0.275 g (50%) of the title compound aswhite crystals.

(ii) EtOOC-CO-(R)Cha-Pro-Agm×HOAc

Prepared by using the deprotection procedure (b) on the product (i)above.

¹ H-NMR (300 MHz, MeOD): δ0.9-2.25 (m, 24H; thereof 1.17 (t, 3H)) 1.90(s, acetate), 3.1-3.25 (m, 4H), 3.5-3.65 (m, 3H; thereof 3.59 (q,2H)),3.88 (m, 1H), 4.35 (m, 1H), 4.69 (dd, 1H).

¹³ C-NMR (75.5 MHz, MeOD): guanidine: δ157.56 and carbonyl carbons:δ159.21, 160.74, 172.81, 174.56.

Example 17 (R,S)Bla-(R)Cha-Pro-Agm×2 TFA

Alkylation as in Example 4 using H-(R)Cha-Pro-Agm(Z) (See Example 3) andα-bromo butyrolacton followed by deprotection procedure (a) gave thetitle compound as a mixture of two diastereomers.

¹ H-NMR (500 MHz, D₂ O, mixture of diastereomers ca: 1/1): δ0.93-1.06(m, 2H), 1.09-1.30 (m, 3H), 1.37-1.49 (m, 1H), 1.50-1.87 (m, 11H),1.89-2.10 (m, 3H), 2.24-2.36 (m, 1H), 2.44-2.56 (m, 1H), 2.72-2.85 (m,1H), 3.10-3.30 (m, 4H), 3.56-3.65 (m, 1H), 3.75-3.84 (m, 1H), 4.2-5.0(m, 5H, partially hidden by the H-O-D signal).

¹³ C-NMR (125.76 MHz, D₂ O) guanidine: δ157.34 (peaks overlapping);carbonyl carbons: δ174.34, 173.90, 173.62, 167.88, 167.58 (two peaks areoverlapping).

Example 18 HOOC-(RorS)CH(CH₂ CH₂ Ph)-(R)Cha-Pro-Agm/b×2 TFA

The title compound was obtained by treating the diastereomer in Example13 by the same way as described in Example 14. This diastereomer cameout after the first one from the column.

¹ H-NMR (500 MHz, MeOD): δ0.95-1.06 (m, 2H), 1.14-1.40 (m, 4H),1.48-1.84 (m, 11H), 1.87-2.30 (m, 6H), 2.72-2.90 (m, 2H), 3.12-3.32 (m,4H), 3.52 (m, 1H), 3.72 (m, 1H), 4.04 (dd, 1H), 4.27 (t, 1H), 4.37 (dd,1H), 7.17-7.32 (m, 5H).

¹³ C-NMR (125 MHz, MeOD): guanidine: δ158.68; carbonyl carbons: δ168.14,171.46, 174.03.

Example 19 H-(R)Cha-Pro-Nag×2 HOAc

(i) Z-(R)Cha-Pro-NH-(CH₂)₃ -NH(Boc)

To a solution of Z-(R)Cha-Pro-OSu (1 mmol) in 1 ml of DMF at 0° C. wasadded H₂ N--(CH₂)₃ --NH(Boc) (See Preparation of starting material)dissolved in 1 ml of DMF and the pH was adjusted to ca: 9 with NMM. Thereaction was stirred at room temperature for 3 days after which it waspoured out on water. The aqueous phase was extracted four times withEtOAc. The combined organic phase was washed twice with 0.3M KHSO₄, 0.2MNaOH, brine and dried. Evaporation and flash chromathography(EtOAc/petroleum ether, 4/1) gave the title compound in 59% yield.

(ii) Z-(R)Cha-Pro-NH-(CH₂)₃ -NH₂

Z-(R)Cha-Pro-NH-(CH₂)₃ --NH(Boc) (0.6 mmol) was dissolved in CH₂ Cl₂ (8ml). TFA (2 ml) was added and the reaction mixture was stirred for 1 h.The solvent was evaporated and the residue was dissolved in CH₂ Cl₂,washed twice with 0.2M NaOH and dried (Na₂ SO₄). Evaporation of thesolvent gave the amine in 93% yield.

¹ H-NMR (500 MHz, CDCl₃) δ0.79-1.03 (m, 2H), 1.05-1.75 (m, 15H),1.84-2.08 (m, 4H), 2.36 (m, 1H), 2.66 (m, 2H), 3.25 (m, 2H), 3.43 (q,1H), 3.85 (m, 1H), 4.45 (m, 1H), 4.56 (d, 1H) 5.09 (m, 2H), 5.35 (d,1H), 7.30-7.45 (m, 5H).

(iii) Z-(R)Cha-Pro-Nag×HOAc

Z-(R)Cha-Pro-NH-(CH₂)₃ -NH₂ (0.55 mmol, 1 eq) was dissolved in DMF (2ml) and the pH adjusted with triethylamine to 8-9.3,5-Dimethyl-1-pyrazolylformamidinium nitrate (0.55 mmol, 1 eq)dissolved in DMF (1 ml) was added and the reaction mixture stirred atroom temperature for three days. The solvent was evaporated, the crudeproduct freeze-dried (H₂ O) and purified with RPLC (CH₃ CN/NH₄ OAc(0.1M), 4/6) to give the title compound in 93% yield after evaporationof the solvent and freeze-drying (H₂ O).

(iv) H-(R)Cha-Pro-Nag×2 HOAc

Prepared by using the deprotection procedure (a) on the product (iii)above.

¹ H-NMR (500 MHz, D₂ O): δ0.82-1.03 (m, 2H), 1.03-1.28 (m, 3H) 1.35 (m,1H), 1.53-1.82 (m, 9H), 1.82-2.05 (m, 3H) 1.89 (s, acetate), 2.24 (m,1H), 3.15 (t, 2H), 3.23 (q, 2H), 3.55 (m, 1H), 3.72 (m, 1H), 4.27-4.34(m, 2H).

¹³ C-NMR (125 MHz, D₂ O): guanidine: δ157.37; carbonyl carbons: δ169.81,174.52.

Example 20 ^(n) Bu-(R)Cha-Pro-Nag×2 HOAc

(i) H-(R)Cha-Pro-Nag(Z)

Prepared from Boc-(R)Cha-Pro-OSu and Boc-Nag(Z) in the same way asdescribed for H-(R)Cha-Pro-Agm(Z) in Example 3.

¹ H-NMR (500 MHz, CDCl₃): δ0.8-1.03 (m, 2H), 1.10-1.50 (m, 6H),1.60-1.83 (m, 8H), 1.87-2.20 (m, 3H), 3.15 (m, 1H), 3.25 (m, 2H), 3.42(m, 2H), 3.63 (dd, H), 3.70 (m, 1H), 4.36 (bs, 1H), 5.07 (s, 2H),7.22-7.43 (m, 5H).

(ii) ^(n) Bu-(R)Cha-Pro-Nag(Z)

H-(R)Cha-Pro-Nag(Z) (0.5 g, 1 mmol) was dissolved in methanol (10 ml).Triethylammonium hydrochloride (0.1 g, 1 mmol), sodium cyanoborohydride(44 mg, 0.7 mmol) and thereafter butyric aldehyde (76 mg, 1.05 mmol)were added and the reaction mixture stirred at room temperature for 20h. The solvent was evaporated and the crude product was dissolved inethyl acetate, washed twice with water, once with brine and dried oversodium sulphate. The solvent was evaporated and the crude product waspurified by flash chromatography (EtOAc/EtOH/Et₃ N, 88/10/2). Finallythe solvent was evaporated and the product dried in vacuo to yield 0.22g (40%) of ^(n) Bu-(R)Cha-Pro-Nag(Z).

¹ H-NMR (500 MHz, CDCl₃): δ0.82-1.0 (m, 5H; thereof 0.88 (t, 3H)),1.08-1.49(m, 1OH), 1.58-1.8 (m, 7H), 1.88-2.22 (m, 3H), 2.4 (m, 1H), 2.5(m, 1H), 3.05 (bs, 1H), 3.3 (m, 1H), 3.4-3.53 (m, 3H), 3.73 (m, 1H),4.42 (bs, 1H), 5.1 (s, 2H), 7.25-7.43 (m, 5H).

(iii) ^(n) Bu-(R)Cha-Pro-Nag×2 HOAc

Prepared by using the deprotection procedure (a) on the product (ii)above.

¹ H-NMR (300 MHz, D₂ O): δ0.94 (t, 2H), 1.10-1.31 (m, 3H), 1.38 (m, 3H),1.55-1.88 (m, 11H), 1.88-2.15 (m, 3H) 1.95 (s, acetate), 2.34 (m, 1H),2.95 (m, 1H), 3.08 (m, 1H), 3.24 (t, 2H), 3.30 (m, 2H), 3.66 (m, 1H),3.82 (m, 1H), 4.32 (t, 1H), 4.41 (dd, 1H).

¹³ C-NMR (125 MHz, D₂ O): guanidine: δ157.40; carbonyl carbons: δ180.39,174.28, 168.55.

Example 21 HO-(CH₂)₃ -(R)Cha-Pro-Nag×2 TFA

(i) BnO-(CH₂)₃ -(R)Cha-Pro-Nag(Z)

1-Benzyloxy 3-trifluoromethanesulfonylpropane (See Prep. of StartingMaterials) (0.5 g, 1 mmol) and H-(R)Cha-Pro-Nag(Z) (See Example 20) weredissolved in tetrahydrofurane (10 ml). Potassium carbonate (0.28 g, 2mmol) was added and the reaction mixture was stirred at room temperaturefor two hours. The solvent was evaporated and the crude productextracted with ethyl acetate/water. The organic phase was washed oncewith aqueous sodium hydrogen carbonate, once with water and once withbrine. After drying over sodium sulphate the solvent was evaporated andthe crude product flash chromatographed (CH₂ CH₂ /MeOH(NH₃ -saturated),95:5). Finally the solvent was evaporated and the product dried in vacuoto yield 0.29 g (45%) of the title compound.

¹ H-NMR (500 MHz, CDCl₃): δ0.77-1.03 (m, 2H), 1.03-2.18 (m, 19H), 2.52(m, 1H), 2.64 (m, 1H), 3.03 (bs, 1H), 3.1-3.6 (m, 7H), 3.66 (m, 1H),4.41 (bs, 1H), 4.46 (s, 2H), 5.08 (s, 2H), 7.2-7.4 (m, 5H), 7.55 (m,1H).

(ii) HO-(CH₂)₃ -(R)Cha-Pro-Nag×2 TFA

Prepared by using the deprotection procedure (a) on the product (i)above.

¹ H-NMR (500 MHz, D₂ O): δ1.00 (bs, 2H), 1.10-1.32 (m, 3H), 1.40 (bs,1H), 1.55-2.15 (m, 14H), 2.30 (m, 1H), 3.05-3.35 (m, 6H), 3.57-3.75 (m,3H), 3.81 (bs, 1H), 4.35 (bs, 1H), 4.42 (bs, 1H).

Example 22 HOOC-CH₂ -(R)Cha-Pro-Nag×HOAc

(i) H-(R)Cha-Pro-NH-(CH₂)₃ -N₃

Prepared in the same way as H-(R)Cha-Pro-Agm(Z) (See Example 3) startingfrom Boc-(R)Cha-Pro-OSu and Boc-NH-(CH₂)₃ -N₃ (replacing Boc-Agm(Z)).

(ii) EtOOC-CH₂ -(R)Cha-Pro-NH-(CH₂)₃ -NH₂ ×HOAc

Alkylation as in Example 4 using H-(R)Cha-Pro-NH-(CH₂)₃ -N₃ andEtOOC--CH₂ --Br followed by deprotection procedure (a) to reduce theazide gave the title compound.

(iii) EtOOC-CH₂ -(R)Cha-Pro-Nag×HOAc

The same procedure as described in Example 19 (iii) for Z-(R)Cha-Pro-Nagwas used to accomplish the guanidation of the amine from (ii) above. Thetitle compound was obtained in a pure form after RPLC (CH₃ CN/NH₄ OAc(0.1M), 3/7) evaporation of the solvent and freeze drying (H₂ O).

(iv) HOOC-CH₂ -(R)Cha-Pro-Nag×HOAc

Prepared by using the deprotection procedure (e) on the product (iii)above.

¹ H-NMR (500 MHz, D₂ O): δ0.99 (m, 2H), 1.09-1.30 (m, 3H), 1.44 (m, 1H),1.59-2.09 (m, 12H) 1.92 (s, acetate), 2.29 (m, 1H), 3.20 (t, 2H), 3.28(m, 2H), 3.52-3.63 (m, 3H), 3.76 (m, 1H), 4.38 (dd, 1H), 4.42 (t, 1H).

¹³ C-NMR (125 MHz, D₂ O): guanidine: δ157.43; carbonyl carbons: δ168.72,171.36, 174.35.

Example 23 EtOOC-CH₂ -(R)Cha-Pro-Nag×HOAc

Prepared according to example 22 (iii).

¹ H-NMR (300 MHz, D₂ O): δ1.07 (m, 2H), 1.17-1.59 (m, 7H; thereof 1.38(t, 3H)), 1.60-2.24 (m, 12H) 2.04 (s, acetate), 2.39 (m, 1H), 3.31 (t,2H), 3.39 (t, 2H), 3.63-3.90 (m, 4H), 4.12 (t, 1H), 4.36 (q, 2H), 4.46(dd, 1H).

¹³ C-NMR (75 MHz, D₂ O,): guanidine: δ157.37; carbonyl carbons: δ173.73,175.09, 175.70.

Example 24 ^(i) PrOOC-CH₂ -(R)Cha-Pro-Nag×HOAc

Alkylation as in Example 4 using H-(R)Cha-Pro-Nag(Z) (See Example 20)and Br--CH₂ COO^(i) Pr followed by deprotection procedure (b) gave thetitle compound.

¹ H-NMR (500 MHz, MeOD): δ0.85-1.05 (m, 2H), 1.1-2.15 (m, 22H; thereof1.23 (d, 3H), 1.25 (d, 3H)), 1.92 (s, acetate), 2.2 (m, 1H), 3.10-3.35(m, 5H), 3.4 (m, 1H), 3.55 (m, 1H), 3.65-3.8 (m, 2H), 4.28 (dd, 1H),5.03 (m, 1H).

¹³ C-NMR (125 MHz, D₂ O): guanidine: δ157.39; carbonyl carbons: δ170.40,172.00 and 174.50.

Example 25 ^(t) BuOOC-CH₂ -(R)Cha-Pro-Nag×2 TFA

Alkylation as in Example 4 using H-(R)Cha-Pro-Nag(Z) (See Example 20)and Br--CH₂ COO^(t) Bu followed by deprotection procedure (b) gave thetitle compound.

¹ H-NMR (300 MHz, MeOD): δ0.9-1.15 (m, 2H), 1.15-2.15 (m, 25H; thereof1.55 (bs, 9H)), 2.3 (m, 1H), 3.15-3.45 (m, 4H), 3.55 (m, 1H), 3.7-3.95(m, 3H), 4.3-4.4 (m, 2H).

¹³ C-NMR (75 MHz, D₂ O): guanidine: δ157.55; carbonyl carbons: δ166.55,168.13 and 174.33.

Example 26 HOOC-CH₂ -OOC-CH₂ -(R)Cha-Pro-Nag×HOAc

(i) BnOOC-CH₂ -OOC-CH₂ -(R)Cha-Pro-Nag(Z)

H-(R)Cha-Pro-Nag(Z) (See Example 20), 0.20 g (0.40 mmol), was mixed with0.115 g (0.40 mmol) of benzyloxycarbonylmethyl bromoacetate, 55 mg of K₂CO₃ (0.40 mmol) and 5 ml of CH₃ CN. The mixture was stirred at roomtemperature for 6 h. The solvent was evaporated and the crude productchromatographed (CH₂ Cl₂ /MeOH, 9/1) to give 0.20 g (71%) of the desiredcompound after evaporation of the solvent.

(ii) HOOC-CH₂ -OOC-CH₂ -(R)Cha-Pro-Nag×HOAc

Prepared by using the deprotection procedure (a) on the product (i)above.

¹ H-NMR (500 MHz, MeOD): δ0.85-1.1 (m, 2H), 1.1-1.6 (m, 8H), 1.6-2.15(m, 10H) 1.99 (s, acetate), 2.23 (m, 1H), 3.1-3.4 (m, 4H), 3.45-3.65 (m,4H), 3.7-3.9 (m, 3H), 4.34 (m, 1H), 4.48 (dd, 2H).

¹³ C-NMR (125 MHz, MeOD), guanidine: δ158.8; carbonyl carbons: δ176.1,175.2, 174.9, 173.1.

Example 27 H₂ N-CO-CH₂ -(R)Cha-Pro-Nag×HOAc

Alkylation as in Example 4 using H-(R)Cha-Pro-Nag(Z) (See Example 20)and Cl--CH₂ CONH₂, in the presence of a catalytic (10 mol %) amount ofKI in the reaction, followed by deprotection procedure (a) gave thetitle compound.

¹ H-NMR (500 MHz, D₂ O): δ1.02 (m, 2H), 1.12-1.34 (m, 3H), 1.46 (m, 1H),1.61-2.13 (m, 9H) 1.99 (s, acetate), 2.34 (m, 1H), 3.25 (t, 2H), 3.33(t, 2H), 3.60-3.82 (m, 4H), 4.22 (t, 1H), 4.41 (dd, 1H).

¹³ C-NMR (75 MHz, D₂ O): guanidine: δ157.5; carbonyl carbons: δ168.94,169.40, 174.43.

Example 28 HOOC-CH₂ -NH-CO-CH₂ -(R)Cha-Pro-Nag×2 TFA

Alkylation as in Example 4 using H-(R)Cha-Pro-Nag(Z) (See Example 20)and Br--CH₂ CONHCH₂ COOBn (See Prep. of starting materials) followed bydeprotection procedure (a) gave the title compound.

¹ H-NMR (500 MHz, MeOD): δ1.01 (m, 2H), 1.15-1.38 (m, 3H), 1.47 (m, 1H),1.64-2.13 (m, 12H), 2.27 (m, 1H), 3.17-3.26 (m, 3H), 3.37 (m, 1H), 3.51(m, 1H), 3.83 (m, 1H), 3.88 (s, 2H), 3.93-4.06 (m, 2H), 4.35-4.45 (m,2H).

¹³ C-NMR (75 MHz, MeOD): guanidine: δ158.71; carbonyl carbons: δ166.94,168.35, 172.44, 174.17.

Example 29 (HOOC-CH₂)₂ -(R)Cha-Pro-Nag×HOAc

(i) (EtOOC-CH₂)₂ -(R)Cha-Pro-NH-(CH₂)₃ -NH₂ ×HOAc

Alkylation as in Example 4 using H-(R)Cha-Pro-NH-(CH₂)₃ -N₃ (See Example22) and Br--CH₂ COOEt (10 eq. was used to accomplish the dialkylation)followed by deprotection procedure (a) gave the title compound.

(ii) (EtOOC-CH₂)₂ -(R)Cha-Pro-Nag×HOAc

The same procedure as described in Example 19 (iii) for Z-(R)Cha-Pro-Nagwas used to accomplish the guanidation of the amine above. Purificationof the compound was made with RPLC (CH₃ CN/NH₄ OAc (0.1M), 4:6)

(iii) (HOOC-CH₂)₂ -(R)Cha-Pro-Nag×HOAc

The hydrolysis of the ester groups was made according to deprotectionprocedure (e) using a double amount of NaOH. The final compound wasobtained pure after RPLC (CH₃ CN/NH₄ OAc (0.1M), 2:8), evaporation ofthe solvent and freeze drying (H₂ O).

¹ H-NMR (300 MHz, D₂ O): δ0.92-1.49 (m, 6H), 1.60-2.54 (m, 10H) 2.05 (s,acetate), 3.25-3.50 (m, 4H), 3.65-4.03 (m, 6H; thereof 3.95 (s, 4H)),4.49 (m, 1H), 4.71 (m, 1H; partly hidden by the H-O-D peak).

¹³ C-NMR (75 MHz, D₂ O): guanidine: δ157.64; carbonyl carbons: δ168.62,171.39, 174.30.

Example 30 HOOC-CH₂ -(Me)(R)Cha-Pro-Nag×2 TFA

(i) Me-(R)Cha-Pro-Nag(Z)

Prepared from Boc-(Me)(R)Cha-Pro-OSu and Boc-Nag(Z) in the same way asdescribed for H-(R)Cha-Pro-Agm(Z) in Example 3.

(ii) HOOC-CH₂ -(Me)(R)Cha-Pro-Nag×2 TFA

Alkylation as in Example 4 using Me-(R)Cha-Pro-Nag(Z) and Br--CH₂ COOBnfollowed by deprotection procedure (b) gave the title compound.

¹ H-NMR (500 MHz, D₂ O): δ0.8-1.06 (m, 2H), 1.08-1.27 (m, 4H), 1.55-2.10(m, 12H), 2.30 (m, 1H), 3.04 (s, 3H), 3.14-3.33 (m, 4H), 3.63 (m, 1H),3.81 (m, 1H), 4.13 (apparent bs, 2H), 4.38 (br.dd, 1H), 4.56 (bt, 1H).

¹³ C-NMR (125.76 MHz, D₂ O): guanidine: δ157.40; carbonyl carbons:δ174.05, 168.83, 167.44.

Example 31 HOOC-CH₂ -(^(n) Bu)(R)Cha-Pro-Nag×2 TFA

Alkylation as in Example 4 using ^(n) Bu-(R)Cha-Pro-Nag(Z) (See Example20) and Br--CH₂ COOBn followed by deprotection procedure (a) gave thetitle compound.

¹ H-NMR (500 MHz, D₂ O): δ0.78-0.88 (m, 3H), 0.88-1.02 (m, 2H),1.02-1.23 (m, 4H), 1.23-1.38 (m, 2H), 1.45-1.84 (m, 11H), 1.84-2.10 (m,3H), 2.24 (m, 1H), 3.05-3.18 (m, 3H), 3.18-3.38 (m, 3H), 3.57 (m, 1H),3.77 (m, 1H), 4.05-4.25 (m, 2H), 4.32 (m, 1H), 4.50 (m, 1H).

¹³ C-NMR (125 MHz, D₂ O): guanidine: δ159.17; carbonyl carbons: δ175.66,171.13, 169.31.

Example 32 HOOC-(R,S)CH(Me)-(R)Cha-Pro-Nag×HOAc

Alkylation as in Example 4 using H-(R)Cha-Pro-Nag(Z) (See Example 20)and Br--CH(Me)COOBn followed by deprotection procedure (a) gave thetitle compound as a mixture of two diastereomers.

Example 33 HOOC-(RorS)CH(Me)-(R)Cha-Pro-Nag/a×HOAc

Obtained by separating the diastereomers formed in Example 32 using RPLC(CH₃ CN/NH₄ OAc (0.1M), 1/4) followed by evaporation of the solvent.This diastereomer came out first of the two from the column.

¹ H-NMR (300 MHz, D₂ O, 2 rotamers ca: 9:1 ratio): δ0.78 (m, minorrotamer), 1.07 (m, 2H), 1.17-1.42 (m, 3H), 1.48-1.64 (m, 4H; thereof1.56 (d, 3H)), 1.64-1.95 (m, 9H), 1.95-2.20 (m, 3H) 2.00 (s, acetate),2.37 (m, 1H), 3.28 (t, 2H), 3.38 (t, 2H), 3.53 (m, minor rotamer), 3.63(m, 2H), 3.77 (m, 1H), 4.24 (d, minor rotamer), 4.35-4.50 (m, 2H), 4.60(d, minor rotamer).

Example 34 HOOC-(RorS)CH(Me)-(R)Cha-Pro-Nag/b×HOAc

The title compound was obtained by using the same procedure as describedin Example 33 on the compound formed in Example 32. This diastereomercame out after the first one from the column.

¹ H-NMR (300 MHz, D₂ O, 2 rotamers ca: 9:1 ratio): δ0.95 (m, minorrotamer), 1.12 (m, 2H), 1.22-1.40 (m, 3H), 1.40-1.67 (m, 4H; thereof1.60 (d, 3H)), 1.67-2.00 (m, 9H), 2.00-2.25 (m, 3H) 2.03 (s, acetate),2.40 (m, 1H), 3.25-3.48 (m, 4H), 3.66-3.84 (m, 2H), 3.93 (m, 1H), 4.38(m, 1H), 4.50 (m, 1H), 4.93 (m, minor rotamer).

¹³ C-NMR (75.5 MHz, D₂ O): δ157.42; carbonyl carbons: δ168.05, 171.99,174.04.

Example 35 EtOOC-(R,S)CH(Me)-(R)Cha-Pro-Nag×2 TFA

Prepared in the same way as described for Example 22 usingEtOOC--CH(Me)--Br instead of Br--CH₂ --COOEt in the alkylation.

¹ H-NMR (500 MHz, MeOD, 2 diastereomers ca: 2.5:1 ratio and 4 rotamers):δ0.88-2.43 (m, 25H), 3.1-4.55 (m, 11H).

¹³ C-NMR (75 MHz, MeOD): guanidine: δ158.65, carbonyl carbons: δ174.33,170.66, 168.20.

Example 36 HOOC-(RorS)CH(nPr)-(R)Cha-Pro-Nag/a×HOAc

Alkylation as in Example 4 using H-(R)Cha-Pro-Nag(Z) (See Example 20)and Br--CH(^(n) Pr)COOEt and deprotection procedure (e) followed bydeprotection procedure (b) gave HOOC-(R,S)CH(^(n) Pr)-(R)Cha-Pro-Agm.The title compound was obtained by separating the diastereomers (thisdiastereomer came out first of the two from the column) by RPLC (CH₃CN/NH₄ OAc (0.1M), 1/4) and freeze drying (H₂ O) after evaporation ofthe solvent.

¹ H-NMR (500 MHz, MeOD): δ0.85-1.05 (m, 5H; thereof 0.95 (t, 3H)),1.1-2.05 (m, 20H) 1.95 (s, acetate), 2.18 (m, 1H), 3.15-3.3 (m, 4H),3.35 (m, 1H), 3.46 (m, 1H), 3.85 (m, 1H), 4.04 (m, 1H), 4.38 (dd, 1H).

¹³ C-NMR (125 MHz, MeOD): guanidine: δ158.73; carbonyl carbons: δ171.63,174.43 and 176.78.

Example 37 HOOC-(R)CH(CH₂ -OH)-(R)Cha-Pro-Nag×2 TFA

Alkylation as in Example 4 using H-(R)Cha-Pro-Nag(Z) (See Example 20)and Br--(S)CH(CH₂ --OBn)--COOBn followed by deprotection procedure (a)gave the title compound.

¹ H-NMR (300 MHz, D₂ O): δ0.75-1.56 (m, 7H), 1.56-2.30 (m, 11H), 2.40(m, 1H), 3.15-3.55 (m, 4H), 3.55-4.60 (m, 7H).

Example 38 HOOC-(R,S)CH(Ph)-(R)Cha-Pro-Nag×2 TFA

Alkylation as in Example 4 using H-(R)Cha-Pro-Nag(Z) (See Example 20)and Br--CH(Ph)COO^(t) Bu and deprotection procedure (a) followed by (f)gave the title compound as a mixture of two diastereomers.

¹ H-NMR (300 MHz, MeOD): δ0.8-1.1 (m, 2H), 1.1-2.18 (m, 16H), 2.26 (m,1H), 3.04-3.35 (m, 5H), 3.45 (m, 1H), 3.7 (m, 1H), 4.35 (m, 1H), 4.85(s, 1H, one isomer), 5.05 (s, 1H, the other isomer), 7.4-7.6 (m, 5H),7.75 (bt, 1H).

¹³ C-NMR (75 MHz, D₂ O): guanidine: δ158.68; carbonyl carbons: δ174.39,174.15 and 170.5, 170.06 and 168.32, 167.78.

Example 39 HOOC-(S)CH(CH₂ CH₂ Ph)-(R)Cha-Pro-Nag×HOAc

Alkylation as in Example 21 using H-(R)Cha-Pro-Nag(Z) (See Example 20)and TfO--(R)CH(CH₂ CH₂ Ph)--COOEt and deprotection procedure (e)followed by (a) gave the title compound.

¹ H-NMR (300 MHz, MeOD): δ0.77-1.05 (m, 2H), 1.05-1.35 (m, 5H),1.35-2.16 (m, 14H) 1.88 (s, acetate), 2.71 (t, 2H), 3.07-3.53 (m, 7H),3.73 (m, 1H), 4.32 (m, 1H), 7.03-7.25 (m, 5H).

¹³ C-NMR (75 MHz, MeOD): guanidine: δ158.71; carbonyl carbons: δ174.15,177.31, 182.61.

Example 40 HOOC-(R)CH(CH₂ CH₂ Ph)-(R)Cha-Pro-Nag×HOAc

Alkylation as in Example 4 using H-(R)Cha-Pro-Nag(Z) (See Example 20)and Br--CH(CH₂ CH₂ Ph)COOEt followed by deprotection procedure (a) and(e) gave HOOC-(R,S)CH(CH₂ -CH₂ -Ph)-(R)Cha-Pro-Nag. The title compoundwas obtained by separating the two diastereomers with RPLC (CH₃ CN/NH₄OAc (0.1M), 2/3) and freeze drying (H₂ O) after evaporation of thesolvent.

¹ H-NMR (300 MHz, MeOD): δ0.97 (m, 2H), 1.10-1.41 (m, 3H), 1.43-2.30 (m,16H) 1.96 (s, acetate), 2.70 (m, 2H), 3.06-3.26 (m, 3H), 3.28-3.66 (m,3H), 3.84 (m, 1H), 4.14 (bt, 1H), 4.39 (dd, 1H), 7.11-7.28 (m, 5H).

¹³ C-NMR (75 MHz, MeOD): guanidine: δ158.66

Example 41 HOOC-CH₂ -CH₂ -(R)Cha-Pro-Nag×HOAc

(i) EtOOC-CH₂ -CH₂ -(R)Cha-Pro-NH-(CH₂)₃ -NH₂

Alkylation as described in Example 15 using H-(R)Cha-Pro-NH-(CH₂)₃ -N₃instead of H-(R)Cha-Pro-Agm(Z) followed by deprotection procedure (a)gave the title compound.

(ii) Et-OOC-CH₂ -CH₂ -(R)Cha-Pro-Nag×HOAc

Guanidation of the amine above in the same way as described in Example19 for Z-(R)Cha-Pro-Nag gave the title compound (ii).

(iii) HOOC-CH₂ -CH₂ -(R)Cha-Pro-Nag×HOAc

Prepared by using the deprotection procedure (e) on the product (ii)above.

¹ H-NMR (500 MHz, D₂ O): δ1.12 (m, 2H), 1.22-1.48 (m,3H), 1.54 (bs, 1H),1.70-2.37 (m, 12H) 2.14 (s, acetate), 2.53 (m, 1H), 2.70 (bs, 2H), 3.15(tw1H), 3.25-3.55 (m, 5H), 3.75 (m, 1H), 3.93 (m, 1H), 4.43 (t, 1H),4.52 (m, 1H).

Example 42 EtOOC-CH₂ -CH₂ -(R)Cha-Pro-Nag×HOAc

Prepared according to Example 41 (ii).

¹ H-NMR (500 MHz, D₂ O): δ0.97 (m, 2H), 1.11-1.39 (m,7H; thereof 1.30(t,3H)), 1.50 (t, 2H), 1.62-1.76 (m,5H), 1.76-2.14 (m, 5H) 1.93 (s,acetate), 2.29 (m, 1H), 2.62 (t, 2H), 2.77-2.94 (m, 2H), 3.23 (t, 2H),3.32 (t, 2H), 3.60-3.87 (m, 3H), 4.20 (q, 2H), 4.36 (dd, 1H).

¹³ C-NMR, (125 MHz, D₂ O): guanidine: δ157.39; carbonyl carbons:δ182.05, 175.13, 175.02.

Example 43 HOOC-(CH₂)₃ -(R)Cha-Pro-Nag×2 HOAc

(i) Et-OOC-CH═CH-CH₂ -(R)Cha-Pro-Nag(Z)

H-(R)Cha-Pro-Nag(Z) (See Example 20) (1 eq) and ethyl 3-bromocrotonate(1.1 eq) were dissolved in acetonitrile (15 ml/mmol). Potassiumcarbonate was added and the reaction mixture stirred at room temperaturefor 2 h. After filtration and evaporation of the solvent, the crudeproduct was purified by flash chromatography (CH₂ Cl₂ /MeOH). Finallythe solvent was evaporated and product dried in vacuo.

¹ H-NMR (500 MHz, CDCl₃): δ0.73-1.0 (m, 2H), 1.0-1.4 (m, 8H; thereof1.33 (t, 3H)), 1.43-2.15 (m, 12H), 2.96 (bs, 1H), 3.12 (dd, 1H),3.16-3.48 (m, 6H), 3.56 (m, 1H), 4.15 (q, 2H), 4.35 (bs, 1H), 5.03 (s,1H), 6.0 (d, 1H), 6.85 (dt, 1H), 7.05 (bs, 1H), 7.17-7.37 (m, 5H), 7.5(bs, 1H).

(ii) EtOOC-(CH₂)₃ -(R)Cha-Pro-Nag×2 TFA

Prepared by using the deprotection procedure (a) on the product (i)above.

(iii) HOOC-(CH₂)₃ -(R)Cha-Pro-Nag×2 HOAc

Prepared by using the deprotection procedure (e) on the product (ii)above.

¹ H-NMR (500 MHz, D₂ O): δ1.02 (bs, 2H), 1.08-1.32 (m, 3H), 1.42 (bs,1H), 1.55-2.15 (m, 14H) 1.92 (s, acetate), 2.33 (bs, 3H), 3.00 (bs, 1H),3.07 (bs, 1H), 3.18-3.40 (m, 4H), 3.62 (bs, 1H), 3.82 (bs, 1H), 4.33(bs, 1H), 4.40 (bs, 1H).

¹³ C-NMR (125 MHz, D₂ O): guanidine: δ157.42; carbonyl carbons: δ181.87,174.34, 168.64.

Example 44 EtOOC-(CH₂)₃ -(R)Cha-Pro-Nag×2 TFA

Prepared according to Example 43 (ii).

¹ H-NMR (300 MHz, MeOD/D₂ O): δ0.63-1.30 (m, 9H; thereof 1.02 (t, 3H)),1.30-1.97 (m, 14H), 2.06 (bs, 1H), 2.28 (m, 2H), 2.72-3.20 (m, 6H), 3.36(m, 1H), 3.60 (m, 1H), 3.94 (m, 2H), 4.06 (m, 1H), 4.17 (m, 1H).

¹³ C-NMR (75 MHz, MeOD/D₂ O): guanidine: δ158.10; carbonyl carbons:δ175.40, 174.23, 168.54.

Example 45 HOOC-CO-(R)Cha-Pro-Nag×HOAc

(i) EtOOC-CO-(R)Cha-Pro-Nag(z)

H-(R)Cha-Pro-Nag(Z), 0.50 g (0.97 mmol) was dissolved in 0.54 mltriethyl amine and 8 ml of CH₂ Cl₂. Ethyl oxalylchloride, 0.146 g (1.07mmol) dissolved in 2 ml of CH₂ Cl₂ was added while the temperature rosefrom 22°-28° C. and the reaction was stirred at room temperature for 2h. The organic phase was washed twice with water, dried (Na₂ SO₄) andflash chromathographed (EtOAc/EtOH(99%), 9/1) to give 92 mg (15%) of thetitle compound.

(ii) HOOC-CO-(R)Cha-Pro-Nag×HOAc

Using the deprotection procedure (b) followed by (e) gave the titlecompound.

¹ H-NMR (300 MHz, MeOD): δ0.88-1.14 (m, 2H), 1.15-1.5 (m, 4H), 1.5-2.3(m, 13H) 1.9 (s, acetate), 3.1-3.43 (m, 4H), 3.6 (m 1H), 4.05 (m, 1H),4.43 (dd, 1H), 4.5 (m, 1H).

¹³ C-NMR (75 MHz, D₂ O): guanidine: δ157.57; carbonyl carbons: δ165.94,173.95, 174,85 and 181.22.

Example 46 MeOOC-CO-(R)Cha-Pro-Nag×HOAc

(i) MeOOC-CO-(R)Cha-Pro-Nag(Z)

The methyl ester was obtained by transesterification ofEtOOC-CO-(R)Cha-Pro-Nag(Z) (See Example 45) on the column during flashchromatography when EtOAc/MeOH(9:1) was used as eluent. Yield 55%.

(ii) MeOOC-CO-(R)Cha-Pro-Nag×HOAc

Prepared by using the deprotection procedure (b) on the product (i)above.

¹ H-NMR (300 MHz, MeOD): δ0.9-1.1 (m, 2H), 1.1-2.3 (m, 17H) 1.9 (s,acetate), 3.12-3.4 (m, 4H), 3.52-3.67 (m, 2H),3.9 (s, 3H), 4.35 (m, 1H),4.65 (m, 1H).

¹³ C-NMR (75 MHz, D₂ O): guanidine: δ157.52; carbonyl carbons: δ159.11,161.20 173.17 and 174.90.

Example 47 (R,S)Bla-(R)Cha-Pro-Nag×2 TFA

Alkylation as in Example 4 using H-(R)Cha-Pro-Nag(Z) (See Example 20)and α-bromo butyrolacton followed by deprotection procedure (a) gave thetitle compound as a mixture of two diastereomers.

¹ H-NMR (300 MHz, D₂ O, mixture of diastreomers): δ1.0-1.43 (m, 5H),1.45-1.60 (br.s, 1H), 1.64-2.28 (m, 12H), 2.31-2.50 (m, 1H), 2.80-2.98(m, 1H), 3.23-3.46 (m, 4H), 3.66-3.79 (m, 1H), 3.82-3.96 (m, 1H),4.33-5.08 (m, 5H, partially hidden by the H-O-D signal).

Example 48 HOOC-(R,S)CH(CH₂ COOH)-(R)Cha-Pro-Nag×HOAc

(i) BnOOC-(R,S)CH(CH₂ COOBn)-(R)Cha-Pro-Nag(Z)

H-(R)Cha-Pro-Nag(Z) (See Example 20), 0.21 g (0.42 mmol), and 0.12 g(0.42 mmol) of dibenzyl maleate were dissolved in 10 ml of CH₃ CN. Themixture was refluxed over night, evaporated and flash chromatographed(CH₂ Cl₂ /MeOH, 94/6). Evaporation of the solvent gave the desiredcompound in 22% yield.

(ii) HOOC-(R,S)CH(CH₂ COOH)-(R)Cha-Pro-Nag×HOAc

Prepared by using the deprotection procedure (a) on the product (i)above. ¹ H-NMR (500 MHz, MeOD): δ0.9-2.4 (m, 19H), 2.00 (s, acetate)2.7-3.0 (m, 2H), 3.1-3.6 (m, 5H), 3.75-3.9 (m, 2H), 4.2-4.5 (m, 2H).

Example 49 MeOOC-(R,S)CH(CH₂ COOMe)-(R)Cha-Pro-Nag×HOAc

(i) MeOOC-(R,S)CH(CH₂ COOMe)-(R)Cha-Pro-Nag(Z)

H-(R)Cha-Pro-Nag(Z) (See Example 20), 0.21 g (0.42 mmol), and 0.24 g(1.7 mmol) of dimethyl maleate were dissolved in 15 ml of MeOH. Themixture was refluxed over night, evaporated and flash chromatographed(CH₂ Cl₂ /MeOH, 9/1). Evaporation of the solvent gave the desiredcompound in 45% yield.

(ii) MeOOC-(R,S)CH(CH₂ COOMe)-(R)Cha-Pro-Nag×HOAc

Prepared by using the deprotection procedure (c) on the product (i)above.

¹ H-NMR (500 MHz, MeOD): δ0.85-1.1 (m, 2H), 1.15-2.3 (m, 17H), 1.91 (s,acetate), 2.6-2.8 (m, 2H), 3.1-3.5 (m, 5H), 3.5-3.8 (m, 10H; thereof 4singlets 3.66, 3.68, 3.71, 3.73), 4.29 (m, 1H).

Example 50 HOOC-Ph-4-CH₂ -(R)Cha-Pro-Nag×2 TFA

(i) ^(t) BuOOC-Ph-4-CH₂ -(R)Cha-Pro-NH-(CH₂)₃ -N₃

H-(R)Cha-Pro-NH-(CH₂)₃ -N₃ (See Example 22), 0.39 g (1.1 mmol) and 0.33g (1.2 mmol) of tertiarybutyl p-bromomethylbenzoate were dissolved in 10ml of CH₃ CN and 0.19 g (2.4 mmol) of K₂ CO₃ was added. The mixture wasrefluxed over night and evaporated. The crude product was flashchromatographed (CH₂ Cl₂ /MeOH, 92:8) to give 0.50 g (84%) of the titlecompound.

(ii) ^(t) BuOOC-Ph-4-CH₂ -(R)Cha-Pro-NH-(CH₂)₃ -NH₂

To a solution of 0.60 g (1.8 mmol) of bis-phenylthio stannane, 0.20 g(1.8 mmol) of thiophenol and 0.18 g (1.8 mmol) of triethyl amine in 50ml of CH₂ Cl₂ at 0° C. was added 0.50 g (0.92 mmol) of ^(t)BuOOC-Ph-4-CH₂ -(R)Cha-Pro-NH-(CH₂)₃ -N₃. The mixture was stirred at 0°C. for 30 min. and at room temperature for 4 h. It was then diluted withCH₂ Cl₂ and washed with aqueous sodium bicarbonate and subsequently 3times with 2% H₂ O₂. The organic layer was extracted with dilute HCl.The combined acidic water phase was washed with EtOAc and subsequentlymade alkaline with NaOH(aq). The aqueous layer was extracted twice withethyl acetate. The combined organic layer was dried (Na₂ SO₄) andevaporated. Flash chromatography (CH₂ Cl₂ /MeOH(NH₃ -saturated), 8:2)gave 0.12 g (26%) of the title compound.

(iii) HOOC-Ph-4-CH₂ -(R)Cha-Pro-Nag×2 TFA

Guanidation of the amine above in the same way as described in Example19 for Z-(R)Cha-Pro-Nag followed by deprotection procedure (f) gave thetitle compound. ¹ H-NMR (500 MHz, MeOD): δ0.9-1.5 (m, 7H), 1.4-1.9 (m,9H), 1.95-2.1 (m, 2H), 2.16 (m, 1H), 2.32 (m, 1H), 3.2-3.3 (m, 3H), 3.41(pentet, 1H), 3.53 (m, 1H), 3.77 (m, 1H), 4.2-4.3 (m, 3H), 4.42 (dd,1H), 7,15 (d, 2H), 8.10 (d, 2H).

¹³ C-NMR (125 MHz, MeOD), guanidine: δ160.8; carbonyl carbons: δ174.3,168.9, 168.2.

Example 51 (HO)₂ P(O)-CH₂ -(R)Cha-Pro-Nag×HOAc

(EtO)₂ PO-CH₂ -(R)Cha-Pro-Nag(Z) (See Example 53), 60 mg (92 mmol), wasdissolved in 3 ml of CH₃ CN. Trimethylsilyl bromide, 0.15 ml, was addedand the mixture was left at room temperature for 21 h. After evaporationand NMR analysis it was found that some ester remained. The crudematerial was again dissolved in 3 ml of CH₃ CN and 0.15 ml oftrimethylsilyl bromide was added. After 5 h the mixture was evaporatedand purified with RPLC (CH₃ CN/NH₄ OAc (0.1M), 30:70) to give the finalcompound after filtration, evaporation and freeze drying in 8% yield.

¹ H-NMR (500 MHz, MeOD): δ0.8-1.1 (m, 2H), 1.15-1.4 (m, 4H), 1.5-1.9 (m,10H), 1.9-2.1 (m, 4H) 1.96 (s, acetate), 2.20 (m, 1H), 2.95 (m, 1H),3.0-3.2 (m, 3H), 3.4-3.5 (m, 2H), 4.09 (m, 1H), 4.39 (bd, 1H), 4.59 (m,1H).

¹³ C-NMR (125 MHz, MeOD): guanidine: δ158.6; carbonyl carbons: δ174.2,170.6

Example 52 EtO(HO)P(O)-CH₂ -(R)Cha-Pro-Nag×2 HOAc

(i) (EtO)(HO)PO-CH₂ -(R)Cha-Pro-Nag(Z).

(EtO)₂ PO-CH₂ -(R)Cha-Pro-Nag(Z) (See Example 53), 50 mg (77 mmol) wasdissolved in 2 ml of EtOH and 2 ml 2M NaOH. The mixture was stirred overnight and evaporated. The crude material was purified with RPLC (CH₃CN/NH₄ OAc (0.1M), 30:70) to give the title compound after filtrationand evaporation of the solvent.

(ii) (EtO)(HO)PO-CH₂ -(R)Cha-Pro-Nag×2 HOAc

Prepared by using deprotection procedure (c) on the product (i) above.

¹ H-NMR (500 MHz, MeOD): δ0.9-1.1 (m, 2H), 1.15-1.35 (m, 6H; thereof1.28 (t, 3H)), 1.35-1.5 (m, 2H), 1.5-1.6 (m, 1H), 1.65-1.8 (m, 6H),1.9-2.1 (m, 3H) 1.95 (s, acetate), 2.19 (m, 1H), 2.8-3.0 (m, 2H),3.1-3.25 (m, 2H), 3.27 (m, 1H), 3.36 (m, 1H), 3.48 (m, 1H), 3.9-4.05 (m,4H), 4.36 (bd, 1H)

¹³ C-NMR (125 MHz, MeOD): guanidine: δ158.6; carbonyl carbons: δ175.0,174.7

Example 53 (EtO)₂ P(O)-CH₂ -(R)Cha-Pro-Nag×HOAc

(i) (EtO)₂ PO-CH₂ -(R)Cha-Pro-Nag(Z).

H-(R)Cha-Pro-Nag(Z) (See Example 20), 0.2 g (0.40 mmol), was dissolvedin 5 ml of THF and 0.11 g (0.80 mmol) of potassium carbonate and 0.12 g(0.40 mmol) diethyl triflylmethyl-phosphonate were added. The mixturewas stirred at room temperature for 2 h. The reaction was worked up withwater and extraction of the aqueous layer three times with EtOAc. Thecombined organic layer was dried (Na₂ SO₄) and evaporated to yield 0.14g (53%) of the title compound.

(ii) (EtO)₂ PO-CH₂ -(R)Cha-Pro-Nag×HOAc

Prepared by using the deprotection procedure (c) on the product (i)above.

¹ H-NMR (500 MHz, MeOD): δ0.85-1.05 (m, 2H), 1.15-1.3 (m, 5H), 1.34 (t,6H), 1.5-1.85 (m, 8H), 1.9-2.05 (m, 3H) 1.91 (s, acetate), 2.10 (m, 1H),2.22 (m, 1H), 2.90 (dd, 1H), 3.05 (dd, 1H), 3.1-3.3 (m, 3H), 3.42 (m,1H), 3.53 (m, 1H), 3.71 (dd, 1H), 3.82 (m, 1H), 4.1-4.2 (m, 4H), 4.28(dd, 1H).

¹³ C-NMR (125 MHz, MeOD), guanidine: δ158.7; carbonyl carbons: δ176.1,175.1.

Example 54 HOOC-CH₂ -(R)Cha-Pro-Mag×HOAc

(i) H-(R) Cha-Pro-H-(CH₂)₂ -NH(Z)

Prepared from Boc-(R)Cha-Pro-OSu and H₂ N--(CH₂)₂ --NH(Z) in the sameway as described for H-(R)Cha-Pro-Agm(Z) in Example 3.

(ii) EtOOC-CH₂ -(R)Cha-Pro-NH-(CH₂)₂ -NH₂ ×HOAc

Alkylation as in Example 4 followed by deprotection procedure (a) gavethe title compound.

(iii) HOOC-CH₂ -(R)Cha-Pro-Mag×HOAc

Guanidation of the amine above in the same way as described in Example19 for Z-(R)Cha-Pro-Nag followed by deprotection procedure (e) gave thetitle compound after purification by RPLC (CH₃ CN/NH₄ OAc (0.1M), 1/4)and freeze drying(H₂ O).

¹ H-NMR (300 MHz, D₂ O): δ0.90-1.18 (m, 2H), 1.19-1.43 (m, 3H), 1.52 (m,1H), 1.63-2.20 (m, 10H) 2.06 (s, acetate), 2.31-2.47 (m, 1H), 3.44 (m,2H), 3.50 (m, 2H), 3.60-3.75 (m, 3H), 3.85 (m, 1H), 4.46-4.54 (m, 2H).

¹³ C-NMR (75 MHz, D₂ O): guanidine: δ157.82; carbonyl carbons: δ168.80,171.41, 174.81.

Example 55 H-(R,S)Pro(3-Ph)-Pro-Agm×2 TFA

Prepared from Boc-(R,S)Pro(3-Ph)-Pro-OSu (See Prep. of startingmaterials) in the same way as described for H-(R)Cha-Pro-Agm(Z) inExample 3 followed by deprotection procedure (b).

¹ H-NMR (500 MHz, D₂ O, mixture of two diastereomers with unknownrelative stereochemistry): δ1.0-1.8 (m, 7H), 2.0-2.5 (m, 3H), 2.8-4.3(m, 10H), 4.56 (d, 1H, major), 4.90 (d, 1H, major), 7.2-7.5 (m, 5H).

¹³ C-NMR (125.76 MHz, D₂ O): guanidine: δ157.36 (minor and major);carbonyl carbons: δ174.1 (major), 174.0 (minor), 167.8 (major), 167.0(minor).

Example 56 H-(R,S)Pro(3-(trans)Ch)-Pro-Agm×2 TFA

Prepared from Boc-(R,S)Pro(3-(trans)Ch)-Pro-OSu (See Prep. of startingmaterials) in the same way as described for H-(R)Cha-Pro-Agm(Z) inExample 3 followed by deprotection procedure (b).

¹ H-NMR (500 MHz, D₂ O, mixture of two diastereomers, ratio 1.8/1):δ0.95-1.32 (m 5H), 1.35-1.46 (m, 1H), 1.50-1.92 (m, 10H), 1.93-2.15 (m,4H), 2.23-2.43 (m, 2H), 3.15-3.30 (m, 4H), 3.35-3.50 (m, 2H), 3.57-3.68(m, 1H), 3.74-3.82 (m, 1H), 4.34-4.41 (m, 1H), 4.51 (d, 1H, minor), 4.48(d, 1H, major).

¹³ C-NMR (125.76 MHz, D₂ O): guanidine: δ157.36 (minor and major),carbonyl carbons: δ174.34 (major), 174.07 (minor), 168.94 (minor andmajor).

Example 57 HOOC-CH₂ -(R,S)Pro(3-(trans)Ph)-Pro-Agm×2 TFA

(i) H-(R,S)Pro(3-(trans)Ph)-Pro-Agm(Z)

Prepared from Boc-(R,S)Pro(3-(trans)Ph)-Pro-OSu (See Prep. of startingmaterials) in the same way as described for H-(R)Cha-Pro-Agm(Z) inExample 3.

(ii) HOOC-CH₂ -(R,S)Pro(3-(trans)Ph)-Pro-Agm×2 TFA

Alkylation as in Example 4 using Br--CH₂ COOBn followed by deprotectionprocedure (b) gave the title compound as a mixture of two diastereomers.

¹ H-NMR (500 MHz, MeOD, mixture of two diastereomers, ratio ca: 1.1/1):δ1.40-1.80 (m, 6H), 1.85-2.05 (m, 1H), 2.10-2.30 (m, 1H), 2.50-2.65 (m,2H), 3.10-3.40 (m, 6H), 3.50-3.70 (m, 2H), 3.9-4.40 (m, 4H), 4.63 (d,1H, major), 4.67 (d, 1H, minor), 7.30-7.60 (m, 5H).

³ C-NMR (125.76 MHz, D₂ O): guanidine: δ157.52 (both isomers); carbonylcarbons: δ173.87, 173.73, 169.12, 168.94, 167.21, 167.00.

Example 58 HOOC-CH₂ -(R,S)Pro(3-(trans)Ph)-Pro-Nag×2 TFA

(i) H-(R,S)Pro(3-(trans)Ph)-Pro-Nag(Z)

Prepared from Boc-(R,S)Pro(3-(trans)Ph)-Pro-OSu (See Prep. of startingmaterials) and Boc-Nag(Z) in the same way as described forH-(R)Cha-Pro-Agm(Z) in Example 3.

(ii) HOOC-CH₂ -(R,S)Pro(3-(trans)Ph)-Pro-Nag×2 TFA

Alkylation as in Example 4 using Br--CH₂ COOBn followed by deprotectionprocedure (b) gave the title compound as a mixture of two diastereomers.

¹ H-NMR (500MHz, MeOD, mixture of two diastereomers, ratio ca: 1.5/1):δ1.40-1.85 (m, 4H), 1.90-2.00 (m, 1H), 2.10-2.30 (m, 1H), 2.45-2.70 (m,2H), 3.08-3.46 (m, 6H), 3.57-3.70 (m, 2H), 3.90-4.0 (m, 1H), 4.32-4.40(m, 1H), 4.04 and 4.29 (AB-quartet, 2H, major), 4.16 and 4.37(AB-quartet, 2H, minor), 4.60 (d, 1H, major), 4.64 (d, 1H, minor),7.3-7.6 (m, 5H).

¹³ C-NMR (125.76 MHz, D₂ O): guanidine: δ157.48 (both isomers); carbonylcarbons: δ173.90, 173.71, 169.01, 168.94, 167.07 (both isomers).

Example 59 HOOC-CH₂ -(R)Cha-Pic-Agm×2 TFA

(i) H-(R)Cha-Pic-Agm(Z)

Prepared from Boc-(R)Cha-Pic-OSu (See Prep. of starting materials) inthe same way as described for H-(R)Cha-Pro-Agm(Z) in Example 3.

(ii) HOOC-CH₂ -(R)Cha-Pic-Agm×2 TFA

Alkylation as in Example 4 using Br--CH₂ COOBn followed by deprotectionprocedure (a) gave the title compound.

¹ H-NMR (300 MHz, MeOD): δ1.02 (m, 2H), 1.13-2.00 (m, 20H), 2.24 (bd,1H), 3.12-3.45 (m, 5H), 3.71 (bd, 1H), 3.87 (s, 2H), 4.65 (bt, 1H), 5.06(m, 1H).

¹³ C-NMR (75 MHz, D₂ O): guanidine: δ157.47; carbonyl carbons: δ169.42,170.03, 172.71.

Example 60 HOOC-CH₂ -(Me)(R)Cha-(R,S)Pic-Agm×HOAc

(i) Me-(R)Cha-(R,S)Pic-Agm(Z)

Prepared from Boc-(Me)(R)Cha-Pic-OSu in the same way as described forH-(R)Cha-Pro-Agm(Z) in Example 3.

(ii) HOOC-CH2-(Me)(R)Cha-(R,S)Pic-Agm×HOAc

Alkylation as in Example 4 using Br--CH₂ COOBn followed by deprotectionprocedure (b) gave the title compound.

Comment: An epimerization of Pic occured somewhere during the synthesis.

The ¹ H-NMR spectrum is complex consisting of two diastereomers ca: 1:1ratio and rotamers thereof.

¹ H-NMR (500 MHz, MeOD): δ0.75-2.15 (several m, 20H) 1.95 (bs, acetate),2.2-2.7 (6H, two distinct sets of signals are observed in the ratio ofca: 1:1; thereof 2.35 and 2.55 (s, 3H)), 3.0-3.5 (m, 6H), 3.9-4.17 (m,2H; thereof 4.14 (dd)), 4.4-4.5 (m, 1H), 4.97-5.15 (two bdd, 1H).

¹³ C-NMR (75 MHz, D₂ O): guanidine: δ157.50; carbonyl carbons: δ169.65,170.01, 170.54, 172.67, 172.89.

Example 61 HOOC-(R,S)CH(Me)-(R)Cha-Pic-Agm×TFA

Alkylation as in Example 4 using H-(R)Cha-Pic-Agm(Z) (See Example 59)and Br--CH(Me)COOBn followed by deprotection procedure (a) gave thetitle compound as a mixture of two diastereomers.

Example 62 HOOC-(RorS)CH(Me)-(R)Cha-Pic-Agm/a×2 TFA

Obtained by separating the diastereomers formed in Example 61 using RPLC(CH₃ CN/NH₄ OAc (0.1M), 1/3) followed by evaporation of the solvent andfreeze-drying from H₂ O/TFA. This diastereomer came out first of the twofrom the column.

¹ H-NMR (300Mz, D₂ O, 2 rotamers ca: 5:1 ratio): δ0.70 (m, minorrotamer), 0.75-1.0 (m, 2H), 1.0-1.28 (m, 3H), 1.28-1.83 (m, 20H; thereof1.57 (d, 3H)), 2.14 (bd, 1H), 2.92 (t, minor rotamer), 3.03-3.32 (m,5H), 3.59 (bd, 1H), 3.85 (q, minor rotamer), 3.98 (q, 1H), 4.30-4.50 (m,minor rotamer), 4.54 (m, 1H), 4.95 (s. 1H).

¹³ C-NMR, (75 MHz, D₂ O): guanidine: δ157.39; carbonyl carbons: δ172.26(2 carbons), 169.92.

Example 63 HOOC-(RorS)CH(Me)-(R)Cha-Pic-Agm/b×2 TFA

The title compound was obtained by using the same procedure as describedin Example 62 on the compound formed in Example 61. This diastereomercame out after the first one from the column.

¹ H-NMR (500 MHz, D₂ O, 2 rotamers ca: 5:1 ratio): δ0.72 (m, minorrotamer), 0.82 (m, minor rotamer), 0.97 (m, 2H), 1.0-1.23 (m, 3H),1.23-1.40 (m, 2H), 1.40-1.83 (m, 18H; thereof 1.63 (d, 3H)), 2.11 (d,1H), 2.17 (d, minor rotamer), 2.92 (t, minor rotamer), 3.05-3.25 (m,4H), 3.29 (t, 1H), 3.74 (d, 1H), 4.02 (q, 1H), 4.34 (d, minor rotamer),4.41 (dd, minor rotamer), 4.52 (t, 1H), 4.95 (s, 1H).

¹³ C-NMR (125 MHz, D₂ O): guanidine: δ154.68; carbonyl carbons: δ169.81,169.60, 167.36.

Example 64 HOOC-CH2-CH2-(R)Cha-Pic-Agm×2 TFA

Prepared from H-(R)Cha-Pic-Agm(Z) (See Example 59) in the same way asdescribed for HOOC-CH₂ -CH₂ -(R)Cha-Pro-Agm in Example 15 using 1.2 eq.of benzylacrylate instead of 1.1 eq.

¹ H-NMR (500 MHz, D₂ O, 2 rotamers ca: 4:1 ratio): δ0.70-0.90 (m, minorrotamer), 0.90-1.0 (m, 2H), 1.05-1.25 (m, 3H), 1.30-1.45 (m, 2H),1.45-1.85 (m, 15H), 2.1 (bd, 1H), 2.2 (bd, minor rotamer), 2.75 (t, 2H),2.95 (t, minor rotamer),3.1-3.4 (m, 7H), 3.75 (bd, 1H), 4.55 (t, 1H),4.95 (m, 1H).

¹³ C-NMR (75 MHz, D₂ O): guanidine: δ157.48; carbonyl carbons: δ170.10,172.58, 174.75.

Example 65 H-(R)Cha-Pic-Nag×2 TFA

(i) Boc-(R)Cha-Pic-Nag(Z)

(ia) Prepared by starting from Boc-(R)Cha-Pic-OSu by using the sameprocedure as described for Boc-(R)Cha-Pro-Agm(Z) in Example 3.

(ib) Prepared by starting from Boc-(R)Cha-Pic-OH

Diphenylphosphoryl azide (0.432 ml, 2 mmol) was added to a stirredsolution of Boc-(R)Cha-Pic-OH (765 mg, 2 mmol) in 5 ml DMF at -10° C.After 10 minutes H-Nag(Z)×2 HCl (600 mg, 2.1 mmol, see Preparation ofStarting Materials) in 5 ml DMF and triethylamine (615 mg, 4.4 mmol) wasadded. The reaction mixture was kept in an ice bath for 3 h and then atroom temperature for 12 h after which it was poured out in water.Extraction of the water phase with EtOAc followed by drying (MgSO₄) ofthe organic phase and evaporation of the solvent in vacuo gave 1.18 g(96%) of the product as a mixture of diastereomers (Epimers in Pic) in aratio of 97:3 (RS/RR).

(ic) Starting from Boc-(R)Cha-Pic-OH

EDC hydrochloride (4.2 g, 21.9 mmol) was added at -15° C. to a stirredsolution of Boc-(R)Cha-Pic-OH (8 g, 20.9 mmol), DMAP (10.6 g, 88 mmol)and H-Nag-(Z)×2 HCl (6.3 g, 19.5 mmol, see Preparation of StartingMaterials) in acetonitrile. The reaction mixture was allowed to warm upto +15° C. during 16 h. The solvent was removed in vacuo an the residuewas dissolved in ethyl acetate. Washing with water, 0.3M KHSO₄, 0,3MNaHCO₃, water and brine followed by drying (Na₂ SO₄) and evaporation ofthe solvent gave 11.9 g (92.5%) of the product as a mixture ofdiastereomers (Epimers in Pic) in a ratio of 98/2 (RS/RR).

¹ H-NMR (500 MHz, CDCl₃): δ0.85-2.0 (m,29H; thereof 1.40 (bs, 9H)), 2.46(bd, 1H), 3.1-3.4 (m, 5H), 3.92 (bd, 1H), 4.53 (bq, 1H), 5.10 (s, 2H),5.22 (bs, 1H), 5.29 (bd, 1H), 6.7-7.2 (b, 3H), 7.25-7.45 (m, 5H).

¹³ C-NMR (125 MHz, CDCl₃): guanidine δ156.9; carbonyl carbons: δ173.6,170.3, 163.7, 161.7.

(ii) H-(R)Cha-Pic-Nag(Z)

Prepared in the same way as described for H-(R)Cha-Pro-Agm(Z) in Example3, starting from Boc-(R)Cha-Pic-Nag(Z).

¹ H-NMR (500 MHz, CDCl₃): δ0.8-2.0 (m, 22H), 2.24 (bd, 1H), 3.1-3.4 (m,5H), 3.72 (bd, 1H), 3.84 (bq, 1H), 5.05 (bd, 1H), 5.08 (s, 2H), 7.3-7.5(m, 5H).

(iii) H-(R)Cha-Pic-Nag×2 TFA

Prepared by using the deprotection procedure (a) on the product (ii)above.

¹ H-NMR (500 MHz, MeOD): δ0.9-1.1 (m, 2H), 1.2-2.0 (m, 18H), 2.32 (bd,1H), 3.20 (t, 2H), 3.30 (t, 2H), 3.36 (m, 1H), 3.69 (bd, 1H), 4.49 (dd,1H), 5.05 (bd, 1H).

¹³ C-NMR (125 MHz, MeOD): guanidine: δ158.7; carbonyl carbons: δ172.7,171.4

Example 66 Me-(R)Cha-(R,S)Pic-Nag×2 TFA

(i) Me-(R)Cha-(R,S)Pic-Nag(Z)

Prepared in the same way as described for H-(R)Cha-Pro-Agm(Z) in Example3 staring from Boc-(Me) (R)Cha-Pic-OSu and Boc-Nag(Z). An epimerizationof Pic occured during the synthesis and the product was obtained asmixture of two diastereomers.

(ii) Me-(R)Cha-(R,S)Pic-Nag×2 TFA

Prepared by using deprotection procedure (b).

The ¹ H-NMR spectrum is complex consisting of two diastereomers ca: 4:1ratio and rotamers thereof.

¹ H-NMR (500 MHz, MeOD): δ0.8-1.08 (m, 2H), 1.15-2.4 (several m, 19H),2.6-2.75 and 2.9-2.95 (several s, 3H) 3.1-3.6 (several m, 5H), 3.75-4.1(several m, 1H) 4.4-4.7 (several m, 1H), 5.05-5.15 (two dd, 1H).

¹³ C-NMR (125 MHz, D₂ O): guanidine: δ154.84; carbonyl carbons: δ167.60and 169.99.

Example 67 HOOC-CH₂ -(R)Cha-Pic-Nag

(i) BnOOC-CH₂ -(R)Cha-Pic-Nag(Z)

Alkylation as in Example 4 using H-(R)Cha-Pic-Nag(Z) (See Example 65)and Br--CH₂ COOBn gave the title compound.

¹ H-NMR (500 MHz, CDCl₃): δ0.8-1.0 (m, 2H), 1.1-1.7 (m, 19H), 1.79 (bd,1H), 2.3-2.5 (m, 2H; thereof 2.38 (bd, 1H)), 3.00 (bt, 1H), 3.1-3.4 (m,5H; thereof 3.38 (d, 1H)) 3.58 (d, 1H), 3.6-3.7 (m, 2H), 5.06 (dd, 2H),5.07 (s, 2H), 5.16 (bs, 1H), 6.7-7.1 (b, 1H), 7.15 (bs, 1H), 7.2-7.4 (m,10H).

¹³ C-NMR (125 MHz, CDCl₃) guanidine and carbonyl carbons: δ176.0, 173.6,170.8, 163.8, 161.7.

(iia) HOOC-CH₂ -(R)Cha-Pic-Nag×2 HCl

Deprotection procedure (a) followed by purification with RPLC using CH₃CN/0.1M NH₄ OAc, 1/3 as eluent, evaporation at 40°-50° C. and freezedrying gave the title compound as the acetate. Treatment with a 20-foldexcess of hydrochloric acid, evaporation and renewed freeze drying gavethe bis-hydrochloride of the desired compound.

¹ H-NMR (500 MHz, D₂ O, mixture of two rotamers): δ0.7-2.0 (m, 20H),2.17 (bd, 1H), 2.95 (t, minor rotamer), 3.17 (t, 2H), 3.25-3.35 (m, 3H),3.72 (bd, 1H), 3.86 (dd, minor rotamer), 3.90 (s, 2H), 4.72 (t, 1H),4.99 (bs, 1H).

¹³ C-NMR (75 MHz, D₂ O); guanidine δ157.4; carbonyl carbons δ169.9,170.2, 173.0.

(iib) HOOC-CH₂ -(R)Cha-Pic-Nag×2 HBr

BnOOC-CH₂ -(R)Cha-Pic-Nag(Z) was dissolved in ^(i) Pr--OH/H₂ O (95/5)and hydrogenated over 5% Pd/C at atmospheric pressure in the presence ofHBr (2.2 eq.). The catalyst was filtered off and the solvent evaporatedto give a yellow oil (Alternatively, the acid can be added afterhydrogenation and filtration). Crystallisation from ^(i) Pr--OH (orEtOH)/EtOAc (1/1) gave the title compound as a white crystalline powder.

¹ H-NMR (500 MHz, D₂ O, mixture of two rotamers): δ1.15-2.0 (m, 20H),2.30 (bd, 1H), 3.30 (m, 2H), 3.40-3.50 (m, 3H), 3.85-3.90 (m, 1H), 3,95(apparent s, 2H), 4.75-4.85 (m, 1H, partially hidden by the H-O-D line),5.10 (bs, 1H).

¹³ C-NMR (125 MHz, D₂ O): guanidine: δ157.6; carbonyl carbons: δ169.7,170.2, 173.0.

Example 68 MeOOC-CH₂ -(R)Cha-Pic-Nag×2 TFA

The methyl ester MeOOC-CH₂ -(R)Cha-Pic-Nag(Z) was obtained by transesterification of ^(i) PrOOC-CH₂ -(R)Cha-Pic-Nag(Z) (See Example 69) onthe column during flash chromatography when CH₂ Cl₂ /MeOH was used aseluent. The title compound was obtained by the deprotection procedure(a).

¹ H-NMR (500 MHz, MeOD): δ0.95-1.15 (m, 2H), 1.2-1.6 (m, 6H), 1.65-2.0(m, 13H), 2.25 (bd, 1H), 3.21 (t, 2H), 3.30 (t, 2H), 3.37 (m, 1H), 3.71(m, 1H), 3.83 (s, 3H), 3.97 (dd, 2H), 4.67 (bt, 1H), 5.05 (bs, 1H).

¹³ C-NMR (125 MHz, MeOD), guanidine: δ158.0; carbonyl carbons: δ173.0,171.1, 168.3.

Example 69 ^(i) PrOOC-CH₂ -(R)Cha-Pic-Nag×2 TFA

Alkylation as described in Example 4 using H-(R)Cha-Pic-Nag(Z) (SeeExample 65) and Br--CH₂ --COO^(i) Pr followed by deprotection procedure(a) gave the title compound.

¹ H-NMR (500 MHz, MeOD): δ0.95-1.1 (m, 2H), 1.15-1.6 (m, 12H; thereof1.25 (d, 3H), 1.28 (d, 3H)), 1.65-1.95 (m, 12H), 2.28 (bd, 1H), 3.21 (t,2H), 3.30 (t,2H), 3.36 (m, 1H), 3.93 (dd, 2H), 4.67 (t, 1H), 5.04 (bs,1H), 5.11 (pentet, 1H).

¹³ C-NMR (125 MHz, MeOD), guanidine: δ157.9; carbonyl carbons: δ173.1,171.0, 168.3.

Example 70 HOOC-CH₂ -(Me)(R)Cha-(RorS)Pic-Nag/b×2 TFA

Alkylation as described in Example 4 using Me-(R)Cha-(R,S)Pic-Nag(Z)(See Example 66) and Br--CH₂ --COOBn followed by deprotection procedure(b) gave HOOC-CH₂ -(Me)(R)Cha-(R,S)Pic-Nag. The two diastereomers whereseparated by RPLC (CH₃ CN/NH₄ OAc, 1:3) followed by freeze-drying fromH₂ O/TFA. This diastereomer came out last of the two from the column.

¹ H-NMR (500 MHz, MeOD): δ0.9-1.1 (m, 2H), 1.15-1.35 (m, 4H), 1.4-1.55(m, 2H), 1.6-1.85 (m, 12H), 2.3 (m, 1H), 2.85 (s, 3H), 3.15-3.45 (m,5H), 3.65 (bs, 2H), 4.0 (m, 1H), 4.65 (m, 1H), 5.08 (dd, 1H).

¹³ C-NMR (75 MHz, D₂ O): guanidine: δ157.65; carbonyl carbons: δ169.86and 172.48.

Example 71 HOOC-(R,S)CH(Me)-(R)Cha-(R,S)Pic-Nag×2 TFA

Alkylation as described in Example 4 using H-(R)Cha-Pic-Nag(Z) (SeeExample 65) and Br--CH(Me)--COOBn followed by deprotection procedure (a)gave the title compound as a mixture of four diastereomers.

Example 72 HOOC-(RorS)CH(Me)-(R)Cha-(RorS)Pic-Nag/c×2 TFA

Obtained by separating the diastereomers formed in Example 71 using RPLC(CH₃ CN/NH₄ OAc (0.1M), 1/4) followed by evaporation and freeze-dryingfrom H₂ O/TFA. This diastereomer came out as the third one of the fourfrom the column.

¹ H-NMR (300 MHz, D₂ O, 2 rotamers ca: 5:1 ratio): δ0.88 (m, minorrotamer), 0.98-1.63 (m, 7H), 1.63-2.02 (m, 16H; thereof 1.68 (d,3H),2.28 (m, 1H), 3.10 (t, minor rotamer), 3.25-3.50 (m, 5H; thereof 3.33(t,2H) and 3.43 (t, 2H)), 3.82 (bd, 1H), 4.02 (q, 1H), 4.55 (d, minorrotamer), 4.65 (d, minor rotamer), 4.72 (m, 1H), 5.10 (m, 1H).

Example 73 HOOC-(RorS)CH(Me)-(R)Cha-(RorS)Pic-Nag/d×2 TFA

Obtained by separating the diastereomers formed in Example 71 using RPLC(CH₃ CN/NH₄ OAc (0.1M), 1:4) followed by evaporation and freeze-dryingfrom H₂ O/TFA. This diastereomer came out last of the four diastereomersfrom the column.

¹ H-NMR (500 MHz, D₂ O, 2 rotamers ca: 5:1 ratio): δ0.80 (m, minorrotamer), 0.90 (m, minor rotamer), 1.03 (m, 2H), 1.10-1.33 (m, 3H), 1.42(m, 2H), 1.51-1.92 (m, 16H; thereof 1.57 (d, 3H)), 2.18 (d, 1H), 2.24(d, minor rotamer), 2.98 (t, minor rotamer), 3.21 (t, 2H), 3.28-3.40 (m,3H; thereof 3.44 (t, 2H)), 3.82 (d, 1H), 4.02 (q, 1H), 4.42 (d, minorrotamer), 4.50 (t, minor rotamer), 4.62 (t, 1H), 4.67 (s, minorrotamer), 5.03 (s, 1H).

Example 74 HOOC-CH2-CH2-(R)cha-Pic-Nag×2 TFA

Prepared from H-(R)Cha-Pic-Nag(Z) (See Example 65) in the same way asdescribed for HOOC-CH₂ -CH₂ -(R)Cha-Pro-Agm in Example 15 using 1.2 eq.of benzylacrylate instead of 1.1 eq.

1H-NMR (500 MHz, D₂ O, 2 rotamers ca: 4:1 ratio): δ0.7-0.9 (m, minorrotamer), 0.9-1.0 (m, 2H), 1.05-1.3 (m, 3H), 1.3-1.45 (m, 2H), 1.5-1.8(m, 13H), 2.10 (d, 1H), 2.20 (d, minor rotamer), 2.75 (t, 2H), 2.95 (t,minor rotamer), 3.15 (t, 2H), 3.2-3.35 (m, 5H), 3.75 (d, 1H), 4.55 (t,1H), 4.95 (m, 1H).

¹³ C-NMR (75 MHz, D₂ O): guanidine: δ157.57; carbonyl carbons: δ170.16,172.82, 174.75.

Example 75 HOOC-CH₂ -(R)Cha-(R,S)Mor-Agm×2 TFA

(i) H-(R)Cha-Mor-Agm(Z)

Prepared from Boc-(R)Cha-Mor-OSu (See Prep. of starting materials) inthe same way as described for H-(R)Cha-Pro-Agm(Z) in Example 3.

(ii) HOOC-CH₂ -(R)Cha-(R,S)Mor-Agm×2 TFA

Alkylation as in Example 4 using Br--CH₂ COOBn followed by deprotectionprocedure (b) gave the title compound. An epimerization of Mor hadoccured somewhere during the synthesis and a mixture of about 9:1 of twodiastereomers was observed in the final product.

¹ H-NMR (300 MHz, MeOD): δ0.92-1.95 (m, 17H), 3.12-3.39 (m, 4H),3.44-4.05 (m, 7H), 4.37 (d, 1H), 4.63 (m, 1H), 4.79 (bd, 1H).

¹³ C-NMR (75.47 MHz, MeOD): guanidine: δ158.63; carbonyl carbons:δ170.87, 170.82, 169.08 others: 5 69.06, 67.01 (C-O-C).

Example 76 HOOC-CH₂ -(R)Cha-(RorS)Mor-Nag×2 TFA

(i) H-(R)Cha-Mor-Nag(Z)

Prepared from Boc-(R)Cha-Mor-OSu (See Prep. of starting materials) andBoc-Nag(Z) in the same way as described for H-(R)Cha-Pro-Agm(Z) inExample 3.

(ii) HOOC-CH₂ -(R)Cha-(RorS)Mor-Nag×2 TFA

Alkylation as described in Example 4 using Br--CH₂ COOBn followed bydeprotection procedure (b) gave the title compound.

¹ H-NMR (300 MHz, MeOD): δ0.92-1.13 (m, 2H), 1.15-1.42 (m, 3H), 1.50(br.s, 1H), 1.62-1.95 (m, 9H), 3.14-3.40 (m, 4H), 3.46-4.13 (m, 7H),4.41 (d, 1H), 4.63 (m, 1H), 4.80 (br.d, 1H).

¹³ C-NMR (75.47 MHz, MeOD): guanidine: δ158.68; carbonyl carbons:δ171.19, 170.90, 169.46. others: δ68.81, 67.00 (C-O-C).

Example 77 H-(R)Cha-Aze-Nag×2 HOAc

(i) Boc-(R)Cha-Aze-Nag(Z)

Prepared from Boc-(R)Cha-Aze-OH in the same way as described forBoc-(R)Cha-Pic-Nag(Z) according to Example 65 (ic).

(ii) H-(R)Cha-Aze-Nag(Z)

Prepared in the same way as described for H-(R)Cha-Pro-Agm(Z) (SeeExample 3).

(iii) H-(R)Cha-Aze-Nag×2 HOAc

Prepared by using the deprotection procedure (a) on the product (ii)above.

¹ H-NMR (300 MHz, D₂ O): δ0.85-1.10 (m, 2H), 1.10-2.04 (m, 13H) 1.95 (s,acetate), 2.20-2.37 (m, 1H), 2.60-2.82 (m, 1H), 3.15-3.40 (m, 4H),3.96-4.15 (m, 2H), 4.18-4.30 (m, 1H), 4.30-4.42 (m, 1H), signals of aminor rotamer appears at: δ0.70, 3.90 and 5.10.

¹³ C-NMR (75 MHz, D₂ O): guanidine: δ157.39 and carbonyl carbons:δ170.22 and 172.38.

Example 78 HOOC-CH₂ -(R)Cha-Aze-Nag×HOAc

(i) BnOOC-CH₂ -(R)Cha-Aze-Nag(Z)

Prepared from H-(R)Cha-Aze-Nag(Z) (See Example 77) according to theprocedure described in Example 4.

(ii) HOOC-CH₂ -(R)Cha-Aze-Nag×HOAc

Prepared by using the deprotection (a) on the product (i) above.

¹ H-NMR (500 MHz, MeOD): δ0.90-1.10 (m, 2H), 1.15-2.00 (m, 13H) 1.95 (s,acetate), 2.20-2.30 (m, 1H), 2.58-2.70 (m, 1H), 3.17-3.30 (m, 4H),3.35-3.50 (m, 2H), 3.55-3.68 (m, 1H), 4.10-4.20 (m, 1H), 4.30-4.38 (m,1H), 4.65-4.77 (m, 1H), signals of minor rotamer appears at: 5 3.75,3.98, 4.03 and 5.08.

¹³ C-NMR (75 MHz, D₂ O): guanidine: δ157.40 and carbonyl carbons:δ169.16, 171.92 and 172.13.

Example 79 H-(R)Cha-Pro(5-(S)Me)-Nag×2 HCl

(i) Boc-(R)Cha-Pro(5-(S)Me)-Nag(Z)

The same procedure as described for the coupling between Boc-(R)Cha-OHand H-Pic-OEt×HCl (See Preparation of Starting Materials) was used toaccomplish the coupling between Boc-(R)Cha-Pro(5-(S)Me)-OH andH-Nag(Z)×2 HCl.

(ii) H-(R)Cha-Pro(5-(S)Me)-Nag(Z)

The same procedure as described for the synthesis ofH-(R)-Cgl-Pic-Nag(Z) (See Example 84 (ii) was used.

(iii) H-(R)Cha-Pro(5-(S)Me)-Nag×2 HCl

Prepared by using the deprotection procedure (d) on the product (ii)above.

¹ H-NMR (300 MHz, D₂ O): δ1.0-2.3 (m, 21H); thereof 1.47 (d, 3H),2.4-2.55 (m, 1H), 3.3-3.6 (m, 4H), 4.30 (bt, 1H), 4.38 (dd, 1H), 4.47(bt, 1H).

¹³ C-NMR (75 MHz, D₂ O): guanidine: δ157.6 carbonyl carbons: δ174.6,169.6.

Example 80 HOOC-CH₂ -(R)Cha-Pro(5-(S)Me)-Nag×HOAc

Alkylation as in Example 4 using H-(R)Cha-Pro(5-(S)Me)-Nag(Z) (SeeExample 79) and Br--CH₂ --COOBn followed by deprotection procedure (a)gave the title compound.

¹ H-NMR (300 MHz, D₂ O): δ0.9-1.9 (m, 19H); thereof 1.34 (bd, 3H), 1.93(s, acetate), 2.0-2.2 (m, 3H), 2.34 (m, 1H), 3.1-3.5 (m, 7H), 3.97 (m,1H), 4.20 (m, 1H), 4.31 (bt, 1H)

¹³ C-NMR (75 MHz, D₂ O): guanidine: δ157.4.

Example 81 HOOC-CH₂ -(R)Cha-(RorS)Pic(4,5-dehydro)-Nag/b×HOAc

(i) Boc-(R)Cha-(R,S)Pic(4,5-dehydro)-Nag(Z)

Prepared from Boc-(R)Cha-(R,S)Pic(4,5-dehydro)-OH in the same way asdescribed for Boc-(R)Cha-Pic-Nag(Z) (See Example 65 (ic)).

(ii) H-(R)Cha-(R,S)Pic(4,5-dehydro)-Nag(Z)

Prepared in the same way as described for H-(R)Cha-Pro-Agm(Z) (SeeExample 3).

(iii) BnOOC-CH₂ -(R)Cha-(R,S)Pic(4,5-dehydro)-Nag(Z)

Prepared from H-(R)Cha-(R,S)Pic(4,5-dehydro)-Nag(Z) according to theprocedure described in Example 4.

(iv) HOOC-CH₂ -(R)Cha-(RorS)Pic(4,5-dehydro)-Nag/b×HOAc

A mixture of 356 mg (0.539 nmol) of BnOOC-CH₂ -(R)Cha-(R,S)Pic(4,5-dehydro)-Nag(Z), 10.8 mL trifluoroaceticacid and 3.4 mltioanisole was stirred at room temperature for 3.5 h. Water was addedand the mixture was washed twice with CH₂ Cl₂ evaporation of the solventgave HOOC-CH₂ -(R)Cha-(R,S)Pic(4,5-dehydro)-Nag. The title compound wasobtained by separating the diastereomers by RPLC (CH₃ CN/NH₄ OAc (0.1M),3/7) and freeze drying (H₂ O) after evaporation of the solvent. Thediastereomer came out last of the two from the column.

¹ H-NMR (300 MHz, D₂ O) δ0.85-1.95 (m, 15H), 2.50-2.80 (m, 2H), 3.25 (t,2H), 3.35 (t, 2H), 3.55 (bs, 2H), 3.85-4.6 (m, 3H), 4.92 (minorrotamer), 5.30 (d, 1H), 5.85-6.1 (m, 2H),

¹³ C-NMR (75 MHz, D₂ O): guanidine: δ157.59; carbonyl carbons: δ171.46,172.58, 173.03.

Example 82 HOOC-CH₂ -(R)Cha-Pic(4-(S)Me)-Nag×2 HCl

(i) Boc-(R)Cha-Pic(4-(S)Me)-Nag(Z)

Prepared from Boc-(R)Cha-Pic(4-(S)Me)-OH in the same way as describedfor Boc-(R)Cha-Pic-Nag(Z) according to method (ic) in Example 65.

(ii) H-(R)Cha-Pic(4-(S)Me)-Nag(Z)

Prepared in the same way as described for H-(R)Cha-Pro-Agm(Z) (SeeExample 3).

(iii) BnOOC-CH₂ -(R)Cha-Pic(4-(S)Me)-Nag(Z)

Prepared from H-(R)Cha-Pic(4-(S)Me)-Nag(Z) according to the proceduredescribed in Example 4.

(iv) HOOC-CH₂ -(R)Cha-Pic(4-(S)Me)-Nag×2 HCl

Prepared by using the deprotection procedure (d) on the product (iii)above.

¹ H-NMR (500 MHz, D₂ O): δ0.95-2.05 (m, 22H; thereof 1.05 (d, 3H)),2.30-2.38 (bd, 1H), 3.28-3.36 (m, 2H) 3.36-3.50 (m, 3H), 3.85-3.95 (m,1H), 3.98 (s, 2H), 4.70-4.90 (m, 1H; partly hidden behind the HODsignal), 5.22-5.27 (d, 1H), signal of a minor rotamer appears at δ0.93,3.13 and 4.57.

¹³ C-NMR (125 MHz, D₂ O): guanidine: δ157.58; carbonyl carbons: δ170.12,170.32 and 172.82.

Example 83 HOOC-CH₂ -(R)Cha-(R)Pic(4-(R)Me)-Nag×2 HCl

(i) Boc-(R)Cha-(R)Pic(4-(R)Me)-Nag(Z)

Prepared from Boc-(R)Cha-(R)Pic(4-(R)Me)-OSu and Boc-Nag(Z) in the sameway as described for Boc-(R)Cha-Pro-Agm(Z) (See Example 3).

(ii) H-(R)Cha-(R)Pic(4-(R)Me)-Nag(Z)

Prepared in the same way as described for H-(R)Cha-Pro-Agm(Z) (SeeExample 3).

(iii) BnOOC-CH₂ -(R)Cha-(R)Pic(4-(R)Me)-Nag(Z)

Prepared from H-(R)Cha-(R)Pic(4-(R)Me)-Nag(Z) according to the proceduredescribed in Example 4.

(iv) HOOC-CH₂ -(R)Cha-(R)Pic(4-(R)Me)-Nag×2 HCl

Prepared by using the deprotection procedure (d) on the product (iii)above.

¹ H-NMR (500 MHz, D₂ O): δ1.00-2.05 (m, 22H), 2.18-2.26 (bd, 1H),3.28-3.36 (m, 2H), 3.36-3.55 (m, 3H), 3.85-4.05 (m, 3H), 4.70-4.90 (m,1H; partly hidden behind the HOD signal), 5.25-5.30 (d, 1H), signals ofminor rotamer appears at: δ2.40, 2.90, 4.10, 4.42, 4.55 and 5.23.

¹³ C-NMR (125 MHz, D₂ O): guanidine: δ157.56: carbonyl carbons: δ169.69,169.84 and 173.20.

Example 84 HOOC-CH₂ -(R)Cgl-Pic-Nag×2 HCl

(i) Boc-(R)Cgl-Pic-Nag(Z)

Prepared from Boc-(R)Cgl-Pic-OH in the same way as described forBoc-(R)Cha-Pic-Nag(Z) according to method (ic) in Example 65.

1H-NMR (300 MHz, CDCl₃): δ0.9-1.8 (m, 27H), 2.4 (d, 1H), 3.1-3.3 (m,5H), 3.9 (d, 1H), 4.2 (t, 1H), 5.1 (s, 2H), 5.2 (bd, 2H), 6.7-7.4 (m,9H).

(ii) H-(R)Cgl-Pic-Nag(Z)

Gaseous hydrogen chloride was bubbled through a solution ofBoc-(R)Cgl-Pic-Nag(Z) (1.38 g, 2.22 mmol) in ethyl acetate (25 ml).After 10 minutes the solvent was evaporated and the residue wasdissolved in ethyl acetate and 10% Na₂ CO₃. The organic phase wasseparated, washed with brine and dried (MgSO₄). Evaporation of thesolvent gave 1.02 g (92%) of the title compound.

¹ H-NMR (300 MHz, MeOD): δ1.0-1.9 (m, 18H), 2.2-2.3 (m, 1H), 3.2-3.3 (m,5H), 3.6 (d, 1H), 3.8-3.9 (bd, 1H), 4.2 (t, 1H), 4.7-4.8 (bs, 5H), 5.1(s, 2H), 5.2 (s, 1H), 7.2-7.3 (m, 5H).

(iii) BnOOC-CH₂ -(R)Cgl-Pic-Nag(Z)

A solution of the triflate ester of benzyl glycolate (291 mg, 0.98 mmol)in CH₂ Cl₂ (2 ml) was added at -25° C. to a stirred mixture ofH-(R)Cgl-Pic-Nag(Z) (0.52 g, 1.04 mmol) and K₂ CO₃ (494 mg, 3.58 mmol)in acetonitrile (5 ml) and CH₂ Cl₂ (1 ml). The temperature was allowedto reach room temperature during a couple of hours and after 5 days thereaction mixture was diluted with water and extracted with EtOAc andtoluene. Drying of the organic phase (MgSO₄) and concentration of thesolution gave 319 mg (47%) of colorless crystals.

¹ H-NMR (500 MHz, CDCl₃): δ1.0-1.1 (m, 1H), 1.1-1.3 (m, 4H), 1.35-1.6(m, 5H), 1.6-1.85 (m, 8H), 1.8-2.2 (bs, 1H), 2.23-2.5 (m, 2H), 2.9 (t,1H), 3.1-3.5 (m, 6H), 3.6-3.7 (m, 2H), 5.0-5.1 (m, 4H), 5.2 (s, 1H),6.5-7.4 (m, 13H).

(iv) HOOC-CH2-(R)Cgl-Pic-Nag×2 HCl

BnOOC-CH2-(R)Cgl-Pic-Nag(Z) (319 mg, 0.49 mmol) was dissolved by heatingin isopropanol (50 ml) and water (5 ml) and hydrogenated for 24 h over10% Pd/C (228 mg). After filtration and evaporation of the solvent andsusequent dissolution in dilute hydrochloric acid followed by freezedrying, the peptide (223 mg, 91%) was isolated as a white powder.

¹ H-NMR (500 MHz, D₂ O): δ1.1-2.1 (m, 18H) 2.3 (d, 1H), 3.3 (t, 2H), 3.4(t, 3H), 3.85-4.05 (m, 3H), 4.6 (d, 1H), 5.15 (s, 1H).

¹³ C-NMR (75 MHz, D₂ O): guanidine: δ157.43 carbonyl carbons: δ169.2,172.94.

Example 85 H-(R)Hoc-Pro-Nag×2 TFA

(i) Boc-(R)Hoc-Pro-Nag(Z)

Prepared from Boc-(R)Hoc-Pro-OH in the same way as described forBoc-(R)Cha-Pic-Nag(Z) according to Example 65 (ic).

(ii) H-(R)Hoc-Pro-Nag(Z)

Prepared in the same way as described for H-(R)Cha-Pro-Agm(Z) (SeeExample 3).

(iii) H-(R)Hoc-Pro-Nag×TFA

Prepared by using the deprotection procedure (a) on the product (ii)above.

¹ H-NMR (300 MHz, D₂ O): δ0.90-1.05 (m, 2H), 1.16-1.48 (m, 6H),1.48-1.84 (m, 6H), 1.84-2.24 (m, 6H), 2.40 (m, 1H), 3.25-3.45 (m, 4H),3,74 (m, 1H), 3.85 (m, 1H), 4.42 (m, 1H), 4.51 (m, 1H).

Example 86 HOOC-CH₂ -(R)Hoc-Pro-Nag×HOAc

(i) BnOOC-CH₂ -(R)Hoc-Pro-Nag(Z)

Prepared from H-(R)Hoc-Pro-Nag(Z) (See Example 85) according to theprocedure described in Example 4.

(ii) HOOC-CH₂ -(R)Hoc-Pro-Nag×HOAc

Prepared by using the deprotection procedure (a) on the product (i)above.

¹ H-NMR (300 MHz, D₂ O): δ0.76-0.97 (m, 2H), 1.00-1.37 (m, 6H),1.50-2.12 (m, 12H) 1.89 (s, acetate), 2.27 (m, 1H), 3.10-3.33 (m, 4H),3.41 (bs, 2H), 3.61 (m, 1H), 3.77 (m, 1H), 4.12 (m, 1H), 4.37 (m, 1H).

¹³ C-NMR (75 MHz, D₂ O): guanidine: δ157.4; carbonyl carbons: δ170.8,173.9, 174.5.

Example 87 HOOC-CH₂ -(R)Hoc-Pic-Nag×HOAc

(i) Boc-(R)Hoc-Pic-Nag(Z)

Prepared from Boc-(R)Hoc-Pic-OH in the same way as described forBoc-(R)Cha-Pic-Nag(Z) according to method (ic) in Example 65.

(ii) H-(R)Hoc-Pic-Nag(Z)

Prepared in the same way as described for H-(R)Cha-Pro-Agm(Z) (SeeExample 3).

(iii) BnOOC-CH₂ -(R)Hoc-Pic-Nag(Z)

Prepared according to the procedure described in Example 4.

(iv) HOOC-CH₂ -(R)Hoc-Pic-Nag×HOAc

Prepared by using the deprotection procedure (a) on the product (iii)above.

¹ H-NMR (300 MHz, D₂ O): δ0.75-0.95 (m, 2H), 1.00-1.30 (m, 6H),1.30-1.50 (m, 2H), 1.50-1.82 (m, 12H), 1.82-1.95 (bs, acetate), 2.23(bd, 1H), 3.08-3.32 (m, 6H), 3.52 (bs, 2H), 3.77 (bd, 1H), 4.50 (bs,1H), 5.00 (bs, 1H).

Example 88 HOOC-CH₂ -(R)Dph-Pic-Nag×2 HCl

(i) Boc-(R)Dph-Pic-Nag(Z)

Prepared from Boc-(R)Dph-Pic-OH in the same way as described forBoc-(R)Cha-Pic-Nag(Z) (See Example 65 (ic)).

(ii) H-(R)Dph-Pic-Nag(Z)

Prepared in the same way as described for H-(R)Cgl-Pic-Nag(Z) (SeeExample 84 (ii)).

(iii) BnOOC-CH₂ -(R)Dph-Pic-Nag(Z)

Prepared from H-(R)Dph-Pic-Nag(Z) according to the procedure describedin Example 4.

(iv) HOOC-CH₂ -(R)Dph-Pic-Nag×2 HCl

Prepared by using the deprotection procedure (d) on the product (iii)above.

¹ H-NMR (500 MHz, D₂ O): δ0.46 (m, 1H), 1.2-1.35 (m, 2H), 1.45 (m, 1H),1.53 (m, 1H), 1.89 (pentet, 2H), 2.03 (bd, 1H), 3.24 (bt, 1H), 3.29 (t,2H), 3.38 (t, 2H), 3.72 (d, 1H), 3.78 (d, 1H), 3.79 (m, 1H), 4.68 (d,1H), 4.89 (m, 1H), 5.73 (d, 1H), 7.4-7.6 (m, 6H), 7.65 (t, 2H), 7.81 (d,2H).

Example 89 HOOC-CH₂ -(R)Dch-Pic-Nag×HOAc

(i) Boc-(R)Dch-Pic-Nag(Z)

Prepared from Boc-(R)Dch-Pic-OH in the same way as described forBoc-(R)Cha-Pic-Nag(Z) (in Example 65 (ic).

(ii) H-(R)Dch-Pic-Nag(Z)

Prepared in the same way as described for H-(R)Cgl-Pic-Nag(Z) (inExample 84 (ii).

(iii) BnOOC-CH₂ -(R)Dch-Pic-Nag(Z)

Prepared from H-(R)Dch-Pic-Nag(Z) according to the procedure describedin Example 4.

(iv) HOOC-CH₂ -(R)Dch-Pic-Nag×HOAc

Prepared by using the deprotection procedure (a) on the product (iii)above.

¹ H-NMR (500 MHz, D₂ O): δ1.2-2.0 (m, 30H), 2.09 (s, acetate), 2.30 (bd,1H), 3.32 (t, 2H), 3.4-3.5 (m, 3H), 3.65 (d, 1H), 3.70 (d, 1H), 3.86(bd, 1H), 4.86 (m, 1H), 5.09 (m, 1H).

¹³ C-NMR (125 MHz, D₂ O): guanidine: δ159.4, carbonyl carbons: δ172.5,173.3, 174.9.

Example P1 Solution for parenteral administration

A solution is prepared from the following ingredients:

    ______________________________________                                        HOOC--CH.sub.2 --(R)Cha-Pic-Nag × 2HBr                                                         5 g                                                    Sodium chloride for injection                                                                        9 g                                                    Acetic acid            3 g                                                    Water for inj. up to 1000 ml                                                  ______________________________________                                    

The active constituent, the sodium chloride and the acetic acid aredissolved in the water. The pH is adjusted with 2M NaOH to pH 3-7. Thesolution is filtered through a sterile 0.2 μm filter and is asepticallyfilled into sterile ampoules.

Example P2 Tablets for oral administration

1000 tablets are prepared from the following ingredients:

    ______________________________________                                        Thrombin inhibitor 100 g                                                      Lactose            200 g                                                      Polyvinyl pyrrolidone                                                                             30 g                                                      Microcrystalline cellulose                                                                        30 g                                                      Magnesium stearate  6 g                                                       ______________________________________                                    

The active constituent and lactose are mixed with an aqueous solution ofpolyvinyl pyrrolidone. The mixture is dried and milled to form granules.The microcrystalline cellulose and then the magnesium stearate are thenadmixed. The mixture is then compressed in a tablet machine giving 1000tablets, each containing 100 mg of active constituent.

Biology

Determination of thrombin clotting time and IC₅₀ TT

Human thrombin (T 6769, Sigma Chem Co) in buffer solution, pH 7.4, 100μl, and inhibitor solution, 100 μl, were incubated for one min. Poolednormal citrated human plasma, 100 μl, was then added and the clottingtime measured in an automatic device (KC 10, Amelung).

The clotting time in seconds was plotted against the inhibitorconcentration, and the IC₅₀ TT was determined by interpolation.

IC₅₀ TT is the concentration of inhibitor that doubles the thrombinclotting time for human plasma. pIC₅₀ TT is the -log 10 of IC₅₀ TT inmol/l. The preferred compounds of the invention have an pIC₅₀ TT in therange 6.6-8.2.

Determination of Activated Partial Thromboplastin Time (APTT)

APTT was determined in pooled normal human citrated plasma with thereagent PTT Automated 5 manufactured by Stago. The inhibitors were addedto the plasma (10 μl inhibitor solution to 90 μl plasma) and APTT wasdetermined in the mixture by use of the coagulation analyser KC10(Amelung) according to the instructions of the reagent producer. Theclotting time in seconds was plotted against the inhibitor concentrationin plasma and the IC₅₀ APTT was determined by interpolation.

IC₅₀ APTT is defined as the concentration of inhibitor in plasma thatdoubled the Activated Partial Thromboplastin Time. pIC₅₀ APTT is the-log 10 of IC₅₀ APTT in mol/l. Those of the preferred compounds of theinvention that were tested showed a pIC₅₀ APTT of 5.1-6.4.

    ______________________________________                                        ABBREVIATIONS                                                                 ______________________________________                                        Agm =      Agmatine                                                           Agm(Z) =   ω-N-benzyloxycarbonyl agmatine                               AA.sub.1 = Amino acid 1                                                       AA.sub.2 = Amino acid 2                                                       Aze =      (S)-Azetidin-2-carboxylic acid                                     Bla =      α-substituted butyrolactone                                  Boc =      tertiary butoxy carbonyl                                           Brine =    saturated water/NaCl solution                                      Bu =       butyl                                                              Bn =       benzyl                                                             Cgl =      (S)-Cyclohexyl glycine                                             Ch =       cyclohexyl                                                         Cha =      (S)-β-cyclohexyl alanine                                      CME-CDI =  1-Cyclohexyl-3-(2-morpholinoethyl)                                            carbodiimide netho-p-toluenesulfonate                              DCC =      dicyclohexyl carbodiimide                                          Dch =      (S)-Dicyclohexyl alanine                                           DMAP =     N,N-diimethyl amino pyridine                                       DMF =      dimethyl formamide                                                 DMSO =     dimethyl sulphoxide                                                Dph =      (S)-Diphenyl alanine                                               EDC =      1-(3-Dimetylaminopropyl)-3-ethylcarbodiimide                                  hydrochloride                                                      Et =       ethyl                                                              EtOAc =    ethyl acetate                                                      HOAc =     acetic acid                                                        HOBt =     N-hydroxy benzotriazole                                            Hoc =      (S)-Homocyclohexyl alanine                                         Hop =      (S)-Homophenyl alanine                                             HOSu =     N-hydroxysuccinimide                                               Mag =      miniagmatine                                                       Me =       methyl                                                             Mor =      (S)-morpholine-2-carboxylic acid                                   Mpa =      mega pascal                                                        Nag =      noragmatine                                                        Nag (Z) =  δ-N-benzyloxycarbonyl-noragmatine                            NMM =      N-methyl morpholine                                                Pgl =      (S)-phenyl glycine                                                 Ph =       phenyl                                                             Phe =      (S)-phenyl alanine                                                 Pic =      (S)-pipecolinic acid                                               Pr =       propyl                                                             Pro =      (S)-proline                                                        RPLC =     reverse phase high-performance liquid                                         chromatography                                                     Tf =       trifluoromethyl sulphonyl                                          TFA =      trifluoracetic acid                                                THF =      tetrahydrofuran                                                    p-TsOH =   para-toluenesulfonic acid                                          Val =      (S)-valine                                                         Z =        benzyloxy carbonyl                                                 ______________________________________                                    

Prefixes n, s, i and t have their usual meanings: normal, iso, sec andtertiary. ##STR10##

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 1                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AlaAspSerGlyGluGlyAspPheLeuAlaGluGlyGlyGlyValArg                              151015                                                                        GlyProArgVal                                                                  20                                                                            __________________________________________________________________________

We claim:
 1. A method of treatment or prophylaxis of venous thrombosisin a mammal in need of such treatment or prophylaxis, which comprisesadministering to the mammal a therapeutically effective amount ofHOOC-CH₂ -(R)Cha-Pic-Nag or a stereoisomer thereof or a physiologicallyacceptable salt thereof.